Reducing immune inhibition induced by SIGLEC-15

ABSTRACT

The present disclosure relates to a modified cell comprising a polynucleotide encoding a secretable scFv binding SIGLEC-15 and/or encoding a dominant negative form of CD44. In embodiments, the modified cell further comprises an antigen-binding molecule, which for example, is a CAR comprising an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain.

CROSS REFERENCE TO RELATED PATENT APPLICATIONS

This application claims the benefit of U.S. Provisional Application 62/837,932, filed Apr. 24, 2019, U.S. Provisional Application 62/837,920, filed Apr. 24, 2019, and U.S. Provisional Application 62/845,631, filed May 9, 2019, which are hereby incorporated by reference in their entirety.

SEQUENCE LISTING INFORMATION

A computer readable textfile, entitled “Sequence Listing.txt,” created on or about Apr. 22, 2020, with a file size of about 312 KB, contains the sequence listing for this application and is hereby incorporated by reference in its entirety.

TECHNICAL FIELD

The present disclosure relates to compositions of agent-sensing immune cells and uses thereof in the treatment of diseases, including cancer.

BACKGROUND

T cell therapies have demonstrated efficacy and curative potential for treating cancers. For example, Chimeric Antigen Receptor (CAR) T cell therapy has achieved good clinical efficacy in cancer, such as B-ALL/CLL/lymphoma. However, use of these therapies has been limited by the presence of an immunosuppressive microenvironment. The immunosuppressive microenvironment includes immune inhibition on, for example, T cells, which is induced by immune inhibitors such as PD1. PD-1 is a negative coregulatory receptor on T cells and antigen-presenting cells. The PD-L1 is expressed by several cell types (e.g., tumor cells and other tissue cells), and appears to be dynamically regulated by the immune microenvironment. Therefore, there is a need to address immune tolerance induced by these immune inhibitors to improve the efficacy of T cell therapies.

SUMMARY

Embodiments relate to a modified cell comprising a polynucleotide encoding a secretable scFv binding SIGLEC-15 and/or encoding a dominant negative form of CD44. In embodiments, the modified cell comprises an antigen-binding molecule, which, for example, is a CAR comprising an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. Embodiments relate to a fusion protein comprising an scFv binding SIGLEC-15, a linker, an extracellular domain, a transmembrane domain, and a cytoplasmic domain, wherein the transmembrane domain is selected from a group consisting of a transmembrane domain of a receptor of IL15, IL2, IL7, IL6, IL12, IL18, IL21, IL23, IL 33, TNFα, TNFβ, IFNα, and IFNβ, and the cytoplasmic domain is selected from a group consisting of a cytoplasmic domain of receptor of the receptor of IL15, IL2, IL7, IL6, IL12, IL18, IL21, IL23, IL 33, TNFα, TNFβ, IFNα, and IFNβ, and the extracellular domain is selected from a group consisting of an extracellular domain of the receptor of IL15, IL2, IL7, IL6, IL12, IL18, IL21, IL23, IL 33, TNFα, TNFβ, IFNα, and IFNβ. Embodiments also relate to a fusion protein comprising an extracellular domain, a transmembrane domain, and a cytoplasmic domain, the transmembrane domain being or comprising the transmembrane domain of Notch.

This Summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter.

BRIEF DESCRIPTION OF THE DRAWINGS

The Detailed Description is described with reference to the accompanying figures. The use of the same reference numbers in different figures indicates similar or identical items.

FIG. 1 shows a schematic diagram of an exemplary fusion protein.

FIG. 2 shows a schematic diagram of another exemplary fusion protein.

FIG. 3 shows a schematic diagram of an exemplary CAR molecule and a fusion protein.

FIG. 4 shows a schematic diagram of an exemplary CAR molecule and a dominant negative CD44.

FIG. 5 shows schematic diagrams of exemplary constructs of dominant negative CD44.

FIG. 6 shows flow cytometry results of CART cells that have been cultured to Day 5.

FIGS. 7, 8, 9, and 10 show CAR expression and functions of T cells comprising a CAR and T cells comprising a CAR and secretable SIGLEC-15 scFv.

FIG. 11 shows a scheme of administering an agent (e.g., an inhibitor or inducer) in exemplary adoptive cell therapy. Arrows indicate the time when an agent (e.g., inhibitor or inducer) is administered to a subject, for example, at the peak of cytokine release.

FIG. 12 shows a schematic diagram of exemplary structures of membrane proteins of a modified cell and uses thereof.

DETAILED DESCRIPTION

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the disclosure belongs. Although any method and material similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, preferred methods and materials are described. For the purposes of the present disclosure, the following terms are defined below.

The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

By “about” is meant a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.

The term “activation,” as used herein, refers to the state of a cell that has been sufficiently stimulated to induce detectable cellular proliferation. Activation can also be associated with induced cytokine production and detectable effector functions. The term “activated T cells” refers to, among other things, T cells that are undergoing cell division.

The term “antibody” is used in the broadest sense and refers to monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multi-specific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired biological activity or function. The antibodies in the present disclosure may exist in a variety of forms including, for example, polyclonal antibodies; monoclonal antibodies; Fv, Fab, Fab′, and F(ab′)₂ fragments; as well as single chain antibodies and humanized antibodies (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y.; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).

The term “antibody fragments” refers to a portion of a full-length antibody, for example, the antigen-binding or variable region of the antibody. Other examples of antibody fragments include Fab, Fab′, F(ab′)₂, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multi-specific antibodies formed from antibody fragments.

The term “Fv” refers to the minimum antibody fragment which contains a complete antigen-recognition and -binding site. This fragment consists of a dimer of one heavy- and one light-chain variable region domain in tight, non-covalent association. From the folding of these two domains emanates six hypervariable loops (3 loops each from the H and L chain) that contribute amino acid residues for antigen-binding and confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv including only three complementarity determining regions (CDRs) specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site (the dimer).

An “antibody heavy chain,” as used herein, refers to the larger of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations. An “antibody light chain,” as used herein, refers to the smaller of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations. K and A light chains refer to the two major antibody light chain isotypes.

The term “synthetic antibody” refers to an antibody which is generated using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage. The term also includes an antibody which has been generated by the synthesis of a DNA molecule encoding the antibody and the expression of the DNA molecule to obtain the antibody or to obtain an amino acid encoding the antibody. The synthetic DNA is obtained using technology that is available and well known in the art.

The term “antigen” refers to a molecule that provokes an immune response, which may involve either antibody production, or the activation of specific immunologically-competent cells, or both. Antigens include any macromolecule, including all proteins or peptides, or molecules derived from recombinant or genomic DNA. For example, DNA including a nucleotide sequence or a partial nucleotide sequence encoding a protein or peptide that elicits an immune response, and therefore, encodes an “antigen” as the term is used herein. An antigen need not be encoded solely by a full-length nucleotide sequence of a gene. An antigen can be generated, synthesized or derived from a biological sample including a tissue sample, a tumor sample, a cell, or a biological fluid.

The term “anti-tumor effect” as used herein, refers to a biological effect associated with a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, decrease in tumor cell proliferation, decrease in tumor cell survival, an increase in life expectancy of a subject having tumor cells, or amelioration of various physiological symptoms associated with the cancerous condition. An “anti-tumor effect” can also be manifested by the ability of the peptides, polynucleotides, cells, and antibodies in the prevention of the occurrence of tumors in the first place.

The term “auto-antigen” refers to an endogenous antigen mistakenly recognized by the immune system as being foreign. Auto-antigens include cellular proteins, phosphoproteins, cellular surface proteins, cellular lipids, nucleic acids, glycoproteins, including cell surface receptors.

The term “autologous” is used to describe a material derived from a subject that is subsequently re-introduced into the same subject.

The term “allogeneic” is used to describe a graft derived from a different subject of the same species. As an example, a donor subject may be related or unrelated to the recipient subject, but the donor subject has immune system markers that are similar to the recipient subject.

The term “xenogeneic” is used to describe a graft derived from a subject of a different species. As an example, the donor subject is from a different species than a recipient subject, and the donor subject and the recipient subject can be genetically and immunologically incompatible.

The term “cancer” is used to refer to a disease characterized by the rapid and uncontrolled growth of aberrant cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers include breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, and the like.

Throughout this specification, unless the context requires otherwise, the words “comprise,” “includes” and “including” will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.

The phrase “consisting of” is meant to include, and is limited to, whatever follows the phrase “consisting of.” Thus, the phrase “consisting of” indicates that the listed elements are required or mandatory and that no other elements may be present.

The phrase “consisting essentially of” is meant to include any element listed after the phrase and can include other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase “consisting essentially of” indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements.

The terms “complementary” and “complementarity” refer to polynucleotides (i.e., a sequence of nucleotides) related by the base-pairing rules. For example, the sequence “A-G-T,” is complementary to the sequence “T-C-A.” Complementarity may be “partial,” in which only some of the nucleic acids' bases are matched according to the base pairing rules, or there may be “complete” or “total” complementarity between the nucleic acids. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands.

The term “corresponds to” or “corresponding to” refers to (a) a polynucleotide having a nucleotide sequence that is substantially identical or complementary to all or a portion of a reference polynucleotide sequence or encoding an amino acid sequence identical to an amino acid sequence in a peptide or protein, or (b) a peptide or polypeptide having an amino acid sequence that is substantially identical to a sequence of amino acids in a reference peptide or protein.

The term “co-stimulatory ligand,” refers to a molecule on an antigen-presenting cell (e.g., an APC, dendritic cell, B cell, and the like) that specifically binds a cognate co-stimulatory molecule on a T cell, thereby providing a signal which, in addition to the primary signal provided by, for instance, binding of a TCR/CD3 complex with an MHC molecule loaded with peptide, mediates a T cell response, including at least one of proliferation, activation, differentiation, and other cellular responses. A co-stimulatory ligand can include B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible co-stimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3/TR6, ILT3, ILT4, HVEM, a ligand for CD7, an agonist or antibody that binds the Toll ligand receptor, and a ligand that specifically binds with B7-H3. A co-stimulatory ligand also includes, inter alia, an agonist or an antibody that specifically binds with a co-stimulatory molecule present on a T cell, such as CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds CD83.

The term “co-stimulatory molecule” refers to the cognate binding partner on a T cell that specifically binds with a co-stimulatory ligand, thereby mediating a co-stimulatory response by the T cell, such as proliferation. Co-stimulatory molecules include an MHC class I molecule, BTLA, and a Toll-like receptor.

The term “co-stimulatory signal” refers to a signal, which in combination with a primary signal, such as TCR/CD3 ligation, leads to T cell proliferation and/or upregulation or downregulation of key molecules.

The terms “disease” and “condition” may be used interchangeably or may be different in that the particular malady or condition may not have a known causative agent (so that etiology has not yet been worked out), and it is therefore not yet recognized as a disease but only as an undesirable condition or syndrome, wherein a more or less specific set of symptoms have been identified by clinicians. The term “disease” is a state of health of a subject wherein the subject cannot maintain homeostasis, and wherein if the disease is not ameliorated then the subject's health continues to deteriorate. In contrast, a “disorder” in a subject is a state of health in which the animal is able to maintain homeostasis, but in which the animal's state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal's state of health.

The term “effective” refers to adequate to accomplish a desired, expected, or intended result. For example, an “effective amount” in the context of treatment may be an amount of a compound sufficient to produce a therapeutic or prophylactic benefit.

The term “encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as a template for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence (except that a “T” is replaced by a “U”) and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

The term “exogenous” refers to a molecule that does not naturally occur in a wild-type cell or organism but is typically introduced into the cell by molecular biological techniques. Examples of exogenous polynucleotides include vectors, plasmids, and/or man-made nucleic acid constructs encoding the desired protein. With regard to polynucleotides and proteins, the term “endogenous” or “native” refers to naturally-occurring polynucleotide or amino acid sequences that may be found in a given wild-type cell or organism. Also, a particular polynucleotide sequence that is isolated from a first organism and transferred to a second organism by molecular biological techniques is typically considered an “exogenous” polynucleotide or amino acid sequence with respect to the second organism. In specific embodiments, polynucleotide sequences can be “introduced” by molecular biological techniques into a microorganism that already contains such a polynucleotide sequence, for instance, to create one or more additional copies of an otherwise naturally-occurring polynucleotide sequence, and thereby facilitate overexpression of the encoded polypeptide.

The term “expression” refers to the transcription and/or translation of a particular nucleotide sequence driven by its promoter.

The term “expression vector” refers to a vector including a recombinant polynucleotide including expression control (regulatory) sequences operably linked to a nucleotide sequence to be expressed. An expression vector includes sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

The term “homologous” refers to sequence similarity or sequence identity between two polypeptides or between two polynucleotides when a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared ×100. For example, if 6 of 10 of the positions in two sequences are matched or homologous, then the two sequences are 60% homologous. By way of example, the DNA sequences ATTGCC and TATGGC share 50% homology. A comparison is made when two sequences are aligned to give maximum homology.

The term “immunoglobulin” or “Ig,” refers to a class of proteins, which function as antibodies. The five members included in this class of proteins are IgA, IgG, IgM, IgD, and IgE. IgA is the primary antibody that is present in body secretions, such as saliva, tears, breast milk, gastrointestinal secretions and mucus secretions of the respiratory and genitourinary tracts. IgG is the most common circulating antibody. IgM is the main immunoglobulin produced in the primary immune response in most subjects. It is the most efficient immunoglobulin in agglutination, complement fixation, and other antibody responses, and is important in defense against bacteria and viruses. IgD is the immunoglobulin that has no known antibody function but may serve as an antigen receptor. IgE is the immunoglobulin that mediates immediate hypersensitivity by causing the release of mediators from mast cells and basophils upon exposure to the allergen.

The term “isolated” refers to a material that is substantially or essentially free from components that normally accompany it in its native state. The material can be a cell or a macromolecule such as a protein or nucleic acid. For example, an “isolated polynucleotide,” as used herein, refers to a polynucleotide, which has been purified from the sequences which flank it in a naturally-occurring state, e.g., a DNA fragment which has been removed from the sequences that are normally adjacent to the fragment. Alternatively, an “isolated peptide” or an “isolated polypeptide” and the like, as used herein, refer to in vitro isolation and/or purification of a peptide or polypeptide molecule from its natural cellular environment, and from association with other components of the cell.

The term “substantially purified” refers to a material that is substantially free from components that are normally associated with it in its native state. For example, a substantially purified cell refers to a cell that has been separated from other cell types with which it is normally associated in its naturally occurring or native state. In some instances, a population of substantially purified cells refers to a homogenous population of cells. In other instances, this term refers simply to a cell that has been separated from the cells with which they are naturally associated in their natural state. In embodiments, the cells are cultured in vitro. In embodiments, the cells are not cultured in vitro.

In the context of the present disclosure, the following abbreviations for the commonly occurring nucleic acid bases are used. “A” refers to adenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refers to thymidine, and “U” refers to uridine.

Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s).

The term “lentivirus” refers to a genus of the Retroviridae family. Lentiviruses are unique among the retroviruses in being able to infect non-dividing cells; they can deliver a significant amount of genetic information into the DNA of the host cell, so they are one of the most efficient methods of a gene delivery vector. Moreover, the use of lentiviruses enables integration of the genetic information into the host chromosome, resulting in stably transduced genetic information. HIV, SIV, and FIV are all examples of lentiviruses. Vectors derived from lentiviruses offer the means to achieve significant levels of gene transfer in vivo.

The term “modulating,” refers to mediating a detectable increase or decrease in the level of a response in a subject compared with the level of a response in the subject in the absence of a treatment or compound, and/or compared with the level of a response in an otherwise identical but untreated subject. The term encompasses perturbing and/or affecting a native signal or response thereby mediating a beneficial therapeutic response in a subject, preferably, a human.

Nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence, or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.

The term “under transcriptional control” refers to a promoter being operably linked to and in the correct location and orientation in relation to a polynucleotide to control (regulate) the initiation of transcription by RNA polymerase and expression of the polynucleotide.

The term “overexpressed” tumor antigen or “overexpression” of the tumor antigen is intended to indicate an abnormal level of expression of the tumor antigen in a cell from a disease area such as a solid tumor within a specific tissue or organ of the patient relative to the level of expression in a normal cell from that tissue or organ. Patients having a solid tumor or a hematological malignancy characterized by overexpression of the tumor antigen can be determined by standard assays known in the art.

Solid tumors are abnormal masses of tissue that usually do not contain cysts or liquid areas. Solid tumors can be benign or malignant. Different types of solid tumors are named for the type of cells that form them (such as sarcomas, carcinomas, and lymphomas). Examples of solid tumors, such as sarcomas and carcinomas, include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer, lung cancers, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, seminoma, bladder carcinoma, melanoma, and CNS tumors (such as a glioma (such as brainstem glioma and mixed gliomas), glioblastoma (also known as glioblastoma multiforme), astrocytoma, CNS lymphoma, germinoma, medulloblastoma, Schwannoma craniopharyogioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, neuroblastoma, retinoblastoma, and brain metastases).

A solid tumor antigen is an antigen expressed on a solid tumor. In embodiments, solid tumor antigens are also expressed at low levels on healthy tissue. Examples of solid tumor antigens and their related disease tumors are provided in Table 1.

TABLE 1 Solid Tumor antigen Disease tumor PRLR Breast Cancer CLCA1 colorectal Cancer MUC12 colorectal Cancer GUCY2C colorectal Cancer GPR35 colorectal Cancer CR1L Gastric Cancer MUC 17 Gastric Cancer TMPRSS11B esophageal Cancer MUC21 esophageal Cancer TMPRSS11E esophageal Cancer CD207 bladder Cancer SLC30A8 pancreatic Cancer CFC1 pancreatic Cancer SLC12A3 Cervical Cancer SSTR1 Cervical tumor GPR27 Ovary tumor FZD10 Ovary tumor TSHR Thyroid Tumor SIGLEC-15 Urothelial cancer SLC6A3 Renal cancer KISS1R Renal cancer QRFPR Renal cancer: GPR119 Pancreatic cancer CLDN6 Endometrial cancer/Urothelial cancer UPK2 Urothelial cancer (including bladder cancer) ADAM12 Breast cancer, pancreatic cancer and the like SLC45A3 Prostate cancer ACPP Prostate cancer MUC21 Esophageal cancer MUC16 Ovarian cancer MS4A12 Colorectal cancer ALPP Endometrial cancer CEA Colorectal carcinoma EphA2 Glioma FAP Mesotelioma GPC3 Lung squamous cell carcinoma IL13-Rα2 Glioma Mesothelin Metastatic cancer PSMA Prostate cancer ROR1 Breast lung carcinoma VEGFR-II Metastatic cancer GD2 Neuroblastoma FR-α Ovarian carcinoma ErbB2 Carcinomasb EpCAM Carcinomasa EGFRvIII Glioma—Glioblastoma EGFR Glioma—NSCL cancer tMUC 1 Cholangiocarcinoma, Pancreatic cancer, Breast Cancer PSCA pancreas, stomach, or prostate cancer

The term “parenteral administration” of a composition includes, e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), intrasternal injection, or infusion techniques.

The terms “patient,” “subject,” and “individual,” and the like are used interchangeably herein and refer to any human, or animal, amenable to the methods described herein. In certain non-limiting embodiments, the patient, subject, or individual is a human or animal. In embodiments, the term “subject” is intended to include living organisms in which an immune response can be elicited (e.g., mammals). Examples of subjects include humans, and animals, such as dogs, cats, mice, rats, and transgenic species thereof.

A subject in need of treatment or in need thereof includes a subject having a disease, condition, or disorder that needs to be treated. A subject in need thereof also includes a subject that needs treatment for prevention of a disease, condition, or disorder.

The term “polynucleotide” or “nucleic acid” refers to mRNA, RNA, cRNA, rRNA, cDNA or DNA. The term typically refers to a polymeric form of nucleotides of at least 10 bases in length, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide. The term includes all forms of nucleic acids including single and double-stranded forms of nucleic acids.

The terms “polynucleotide variant” and “variant” and the like refer to polynucleotides displaying substantial sequence identity with a reference polynucleotide sequence or polynucleotides that hybridize with a reference sequence under stringent conditions that are defined hereinafter. These terms also encompass polynucleotides that are distinguished from a reference polynucleotide by the addition, deletion or substitution of at least one nucleotide. Accordingly, the terms “polynucleotide variant” and “variant” include polynucleotides in which one or more nucleotides have been added or deleted or replaced with different nucleotides. In this regard, it is well understood in the art that certain alterations inclusive of mutations, additions, deletions, and substitutions can be made to a reference polynucleotide whereby the altered polynucleotide retains the biological function or activity of the reference polynucleotide or has increased activity in relation to the reference polynucleotide (i.e., optimized). Polynucleotide variants include, for example, polynucleotides having at least 50% (and at least 51% to at least 99% and all integer percentages in between, e.g., 90%, 95%, or 98%) sequence identity with a reference polynucleotide sequence described herein. The terms “polynucleotide variant” and “variant” also include naturally-occurring allelic variants and orthologs.

The terms “polypeptide,” “polypeptide fragment,” “peptide,” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues and to variants and synthetic analogues of the same. Thus, these terms apply to amino acid polymers in which one or more amino acid residues are synthetic non-naturally occurring amino acids, such as a chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally-occurring amino acid polymers. In certain aspects, polypeptides may include enzymatic polypeptides, or “enzymes,” which typically catalyze (i.e., increase the rate of) various chemical reactions.

The term “polypeptide variant” refers to polypeptides that are distinguished from a reference polypeptide sequence by the addition, deletion, or substitution of at least one amino acid residue. In embodiments, a polypeptide variant is distinguished from a reference polypeptide by one or more substitutions, which may be conservative or non-conservative. In embodiments, the polypeptide variant comprises conservative substitutions and, in this regard, it is well understood in the art that some amino acids may be changed to others with broadly similar properties without changing the nature of the activity of the polypeptide. Polypeptide variants also encompass polypeptides in which one or more amino acids have been added or deleted or replaced with different amino acid residues.

The term “promoter” refers to a DNA sequence recognized by the synthetic machinery of the cell or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence. The term “expression control (regulatory) sequences” refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

The term “bind,” “binds,” or “interacts with” refers to a molecule recognizing and adhering to a second molecule in a sample or organism but does not substantially recognize or adhere to other structurally unrelated molecules in the sample. The term “specifically binds,” as used herein with respect to an antibody, refers to an antibody which recognizes a specific antigen, but does not substantially recognize or bind other molecules in a sample. For example, an antibody that specifically binds an antigen from one species may also bind that antigen from one or more species. But, such cross-species reactivity does not itself alter the classification of an antibody as specific. In another example, an antibody that specifically binds an antigen may also bind different allelic forms of the antigen. However, such cross reactivity does not itself alter the classification of an antibody as specific. In some instances, the terms “specific binding” or “specifically binding,” can be used in reference to the interaction of an antibody, a protein, or a peptide with a second chemical species, to mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds a specific protein structure rather than to any protein. If an antibody is specific for epitope “A,” the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled “A” and the antibody, will reduce the amount of labeled A bound to the antibody.

By “statistically significant,” it is meant that the result was unlikely to have occurred by chance. Statistical significance can be determined by any method known in the art. Commonly used measures of significance include the p-value, which is the frequency or probability with which the observed event would occur if the null hypothesis were true. If the obtained p-value is smaller than the significance level, then the null hypothesis is rejected. In simple cases, the significance level is defined at a p-value of 0.05 or less. A “decreased” or “reduced” or “lesser” amount is typically a “statistically significant” or a physiologically significant amount, and may include a decrease that is about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, or 50 or more times (e.g., 100, 500, 1000 times) (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.) an amount or level described herein.

The term “stimulation,” refers to a primary response induced by binding of a stimulatory molecule (e.g., a TCR/CD3 complex) with its cognate ligand thereby mediating a signal transduction event, such as signal transduction via the TCR/CD3 complex. Stimulation can mediate altered expression of certain molecules, such as downregulation of TGF-β, and/or reorganization of cytoskeletal structures.

The term “stimulatory molecule” refers to a molecule on a T cell that specifically binds a cognate stimulatory ligand present on an antigen presenting cell. For example, a functional signaling domain derived from a stimulatory molecule is the zeta chain associated with the T cell receptor complex. The stimulatory molecule includes a domain responsible for signal transduction.

The term “stimulatory ligand” refers to a ligand that when present on an antigen presenting cell (e.g., an APC, a dendritic cell, a B-cell, and the like.) can specifically bind with a cognate binding partner (referred to herein as a “stimulatory molecule”) on a cell, for example a T cell, thereby mediating a primary response by the T cell, including activation, initiation of an immune response, proliferation, and similar processes. Stimulatory ligands are well-known in the art and encompass, inter alia, an MHC Class I molecule loaded with a peptide, an anti-CD3 antibody, a superagonist anti-CD28 antibody, and a superagonist anti-CD2 antibody.

The term “therapeutic” refers to a treatment and/or prophylaxis. A therapeutic effect is obtained by suppression, remission, or eradication of a disease state or alleviating the symptoms of a disease state.

The term “therapeutically effective amount” refers to the amount of the subject compound that will elicit the biological or medical response of a tissue, system, or subject that is being sought by the researcher, veterinarian, medical doctor or another clinician. The term “therapeutically effective amount” includes that amount of a compound that, when administered, is sufficient to prevent the development of, or alleviate to some extent, one or more of the signs or symptoms of the disorder or disease being treated. The therapeutically effective amount will vary depending on the compound, the disease and its severity and the age, weight, etc., of the subject to be treated.

The term “treat a disease” refers to the reduction of the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject.

The term “transfected” or “transformed” or “transduced” refers to a process by which an exogenous nucleic acid is transferred or introduced into the host cell. A “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed, or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

The term “vector” refers to a polynucleotide that comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art including linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term “vector” includes an autonomously replicating plasmid or a virus. The term also includes non-plasmid and non-viral compounds which facilitate the transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include adenoviral vectors, adeno-associated virus vectors, retroviral vectors, and others. For example, lentiviruses are complex retroviruses, which, in addition to the common retroviral genes gag, pol, and env, contain other genes with regulatory or structural function. Lentiviral vectors are well known in the art. Some examples of lentivirus include the Human Immunodeficiency Viruses: HIV-1, HIV-2, and the Simian Immunodeficiency Virus: SIV. Lentiviral vectors have been generated by multiply attenuating the HIV virulence genes, for example, the genes env, vif, vpr, vpu, and nef are deleted making the vector biologically safe.

Ranges: throughout this disclosure, various aspects of the disclosure can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

A “chimeric antigen receptor” (CAR) molecule is a recombinant polypeptide including at least an extracellular domain, a transmembrane domain and a cytoplasmic domain or intracellular domain. In embodiments, the domains of the CAR are on the same polypeptide chain, for example a chimeric fusion protein. In embodiments, the domains are on different polypeptide chains, for example the domains are not contiguous.

The extracellular domain of a CAR molecule includes an antigen-binding domain. The antigen-binding domain is for expanding and/or maintaining the modified cells, such as a CAR T cell or for killing a tumor cell, such as a solid tumor. In embodiments, the antigen-binding domain for expanding and/or maintaining modified cells binds an antigen, for example, a cell surface molecule or marker, on the surface of a WBC. In embodiments, the WBC is a granulocyte, monocyte and or lymphocyte. In embodiments, the WBC is a lymphocyte, for example, a B cell. In embodiments, the WBC is a B cell. In embodiments, the cell surface molecule of a B cell includes CD19, CD22, CD20, BCMA, CD5, CD7, CD2, CD16, CD56, CD30, CD14, CD68, CD11b, CD18, CD169, CD1c, CD33, CD38, CD138, or CD13. In embodiments, the cell surface molecule of the B cell is CD19, CD20, CD22, or BCMA. In embodiments, the cell surface molecule of the B cell is CD19.

Modified cells (e.g., T-cells and NK cells) may be derived from a stem cell. The stem cells may be adult stem cells, embryonic stem cells, more particularly non-human stem cells, cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells, totipotent stem cells or hematopoietic stem cells. A modified cell may also be a dendritic cell, a NK-cell, a B-cell or a T-cell selected from the group consisting of inflammatory T-lymphocytes, cytotoxic T-lymphocytes, regulatory T-lymphocytes or helper T-lymphocytes. In another embodiment, Modified cells may be derived from the group consisting of CD4+T-lymphocytes and CD8+T-lymphocytes. Prior to expansion and genetic modification of the cells of the invention, a source of cells may be obtained from a subject through a variety of non-limiting methods. T cells may be obtained from a number of non-limiting sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In embodiments of the present invention, any number of T cell lines available and known to those skilled in the art, may be used. In embodiments, modified cells may be derived from a healthy donor, from a patient diagnosed with cancer or from a patient diagnosed with an infection. In embodiments, modified cell is part of a mixed population of cells which present different phenotypic characteristics.

The term “stem cell” refers to any of certain types of cell which have the capacity for self-renewal and the ability to differentiate into other kind(s) of cell. For example, a stem cell gives rise either to two daughter stem cells (as occurs in vitro with embryonic stem cells in culture) or to one stem cell and a cell that undergoes differentiation (as occurs e.g. in hematopoietic stem cells, which give rise to blood cells). Different categories of stem cell may be distinguished on the basis of their origin and/or on the extent of their capacity for differentiation into other types of cell. For example, stem cell may include embryonic stem (ES) cells (i.e., pluripotent stem cells), somatic stem cells, Induced pluripotent stem cells, and any other types stem cells.

The pluripotent embryonic stem cells may be found in the inner cell mass of a blastocyst and have high innate capacity for differentiation. For example, pluripotent embryonic stem cells may have the potential to form any type of cell in the body. When grown in vitro for long periods of time, ES cells maintain pluripotency: progeny cells retain the potential for multilineage differentiation.

Somatic stem cells may include the fetal stem cells (from the fetus) and adult stem cells (found in various tissues, such as bone marrow). These cells have been regarded as having a capacity for differentiation lower than that of the pluripotent ES cells—with the capacity of fetal stem cells being greater than that of adult stem cells; they apparently differentiate into only a limited range of types of cell and have been described as multipotent. The ‘tissue-specific’ stem cells normally give rise to only one type of cell. For example, embryonic stem cells may be differentiated into blood stem cells (e.g., Hematopoietic stem cells (HSCs)), which may be further differentiated into various blood cells (e.g., red blood cells, platelets, white blood cells, etc.).

Induced pluripotent stem cells (i.e., iPS cells or iPSCs) may include a type of pluripotent stem cell artificially derived from a non-pluripotent cell (e.g., an adult somatic cell) by inducing a expression of specific genes. Induced pluripotent stem cells are similar to natural pluripotent stem cells, such as embryonic stem (ES) cells, in many aspects, such as the expression of certain stem cell genes and proteins, chromatin methylation patterns, doubling time, embryoid body formation, teratoma formation, viable chimera formation, and potency and differentiability. Induced pluripotent cells may be made from adult stomach, liver, skin cells and blood cells.

In embodiments, the antigen-binding domain for killing a tumor, binds an antigen on the surface of a tumor, for example a tumor antigen or tumor marker. Tumor antigens are proteins that are produced by tumor cells that elicit an immune response, particularly T cell mediated immune responses. Tumor antigens are well known in the art and include, for example, tumor associated MUC1 (tMUC1), a glioma-associated antigen, carcinoembryonic antigen (CEA), β-human chorionic gonadotropin, alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxyl esterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostein, PSMA, Her2/neu, surviving, telomerase, prostate-carcinoma tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrinB2, CD22, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, CD19, and mesothelin. For example, when the tumor antigen is CD19, the CAR thereof can be referred to as CD19CAR, which is a CAR molecule that includes a antigen-binding domain that binds CD19.

In embodiments, the extracellular antigen-binding domain of a CAR includes at least one scFv or at least a single domain antibody. As an example, there can be two scFvs on a CAR. The scFv includes a light chain variable (VL) region and a heavy chain variable (VH) region of a target antigen-specific monoclonal antibody joined by a flexible linker. Single chain variable region fragments can be made by linking light and/or heavy chain variable regions by using a short linking peptide (Bird et al., Science 242:423-426, 1988). An example of a linking peptide is the GS linker having the amino acid sequence (GGGGS)₃ (SEQ ID NO: 120), which bridges approximately 3.5 nm between the carboxy terminus of one variable region and the amino terminus of the other variable region. Linkers of other sequences have been designed and used (Bird et al., 1988, supra). In general, linkers can be short, flexible polypeptides and preferably comprised of about 20 or fewer amino acid residues. The single chain variants can be produced either recombinantly or synthetically. For synthetic production of scFv, an automated synthesizer can be used. For recombinant production of scFv, a suitable plasmid containing polynucleotide that encodes the scFv can be introduced into a suitable host cell, either eukaryotic, such as yeast, plant, insect or mammalian cells, or prokaryotic, such as E. coli. Polynucleotides encoding the scFv of interest can be made by routine manipulations such as ligation of polynucleotides. The resultant scFv can be isolated using standard protein purification techniques known in the art.

The cytoplasmic domain of the CAR molecules described herein includes one or more co-stimulatory domains and one or more signaling domains. The co-stimulatory and signaling domains function to transmit the signal and activate molecules, such as T cells, in response to antigen-binding. The one or more co-stimulatory domains are derived from stimulatory molecules and/or co-stimulatory molecules, and the signaling domain is derived from a primary signaling domain, such as the CD3 zeta domain. In embodiments, the signaling domain further includes one or more functional signaling domains derived from a co-stimulatory molecule. In embodiments, the co-stimulatory molecules are cell surface molecules (other than antigens receptors or their ligands) that are required for activating a cellular response to an antigen.

In embodiments, the co-stimulatory domain includes the intracellular domain of CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, or any combination thereof. In embodiments, the signaling domain includes a CD3 zeta domain derived from a T cell receptor.

The CAR molecules described herein also include a transmembrane domain. The incorporation of a transmembrane domain in the CAR molecules stabilizes the molecule. In embodiments, the transmembrane domain of the CAR molecules is the transmembrane domain of a CD28 or 4-1BB molecule.

Between the extracellular domain and the transmembrane domain of the CAR, there may be incorporated a spacer domain. As used herein, the term “spacer domain” generally means any oligo- or polypeptide that functions to link the transmembrane domain to the extracellular domain and/or the cytoplasmic domain on the polypeptide chain. A spacer domain may include up to 300 amino acids, preferably 10 to 100 amino acids, and most preferably 25 to 50 amino acids.

Embodiments relate to a modified cell comprising a binding molecule, and a dominant negative form of an inhibitory immune checkpoint molecule, wherein expression of the dominant negative form of the inhibitory immune checkpoint molecule is regulated by an inducible gene expression system. Embodiments relate to a polynucleotide encoding the binding molecule and/or the dominant negative form of the inhibitory immune checkpoint molecule. Embodiments relate to a pharmaceutical composition comprising the population of the modified cell. Embodiments relate to a kit comprising an effective amount of vector-free nucleic acids encoding the binding molecule and/or the dominant negative form of the inhibitory immune checkpoint molecule to render a population of immune cells specific for a tumor antigen expressed on the surface of the cells of a subject. Embodiments relate to a method of eliciting or enhancing T cell response, treating a subject in need thereof or enhancing cancer treatment thereof, the method comprising administering an effective amount of the composition or the kit to the subject.

Embodiments relate to a pharmaceutical composition comprising a population of the modified cells and a population of additional modified cells, wherein the modified cells bind a first antigen, and the additional modified cells bind a second antigen, which is different form the first antigen. In embodiments, the first antigen is a white blood cell antigen, and the second antigen is a solid tumor antigen. In embodiments, the second antigen is a white blood cell antigen, and the first antigen is a solid tumor antigen. In embodiments, the white blood cell antigen is CD19, CD22, CD20, BCMA, CD5, CD7, CD2, CD16, CD56, CD30, CD14, CD68, CD11b, CD18, CD169, CD1c, CD33, CD38, CD138, or CD13. In embodiments, the blood cell antigen is CD19, CD20, CD22, or BCMA. In embodiments, the solid tumor antigen is tMUC 1, PRLR, CLCA1, MUC12, GUCY2C, GPR35, CR1L, MUC 17, TMPRSS11B, MUC21, TMPRSS11E, CD207, SLC30A8, CFC1, SLC12A3, SSTR1, GPR27, FZD10, TSHR, SIGLEC-15, SLC6A3, KISS1R, CLDN18.2, QRFPR, GPR119, CLDN6, UPK2, ADAM12, SLC45A3, ACPP, MUC21, MUC16, MS4A12, ALPP, CEA, EphA2, FAP, GPC3, IL13-Rα2, Mesothelin, PSMA, ROR1, VEGFR-II, GD2, FR-α, ErbB2, EpCAM, EGFRvIII, B7-H3, or EGFR. In embodiments, the solid tumor antigen comprises tumor associated MUC1, ACPP, TSHR, GUCY2C, UPK2, CLDN18.2, PSMA, DPEP3, CXCRS, B7-H3, MUC16, CLDN6, Muc17, PRLR, and FZD10.

Embodiments relate to a method or use of polynucleotide, the method comprising providing a viral particle (e.g., AAV, lentivirus or their variants) comprising a vector genome, the vector genome comprising the polynucleotide encoding the binding molecule and/or the dominant negative form of the inhibitory immune checkpoint molecule, the polynucleotide operably linked to an expression control element conferring transcription of the polynucleotides; and administering an amount of the viral particle to a subject such that the polynucleotide is expressed in the subject, where the one or more molecules are overexpressed in cancer cells, associated with recruitment of immune cells, and/or associated with autoimmunity. In embodiments, the AAV preparation may include AAV vector particles, empty capsids and host cell impurities, thereby providing an AAV product substantially free of AAV empty capsids.

In embodiments, expression of the one or more molecules may be regulated by an inducible expression system. The inducible expression system allows for a temporal and spatial controlled activation and/or expression of genes. For example, Tetracycline-Controlled Transcriptional Activation is a method of inducible gene expression where transcription is reversibly turned on or off in the presence of the antibiotic tetracycline or one of its derivatives (e.g., doxycycline). For example, an inducible suicide gene expression system allows for a temporal and spatial controlled activation and/or expression of a suicide gene, which causes a cell to kill itself through apoptosis. In embodiments, the modified cells comprise a nucleic acid sequence encoding a reverse tetracycline transactivator (rtTA). In embodiments, expression of the one or more molecules is regulated by the rtTA, such that the one or more molecules are expressed in the presence of tetracycline. In embodiments, a concentration of tetracycline in the cell culture medium is not less than about 2 μg/ml. In embodiments, the tetracycline is selected from the group consisting of tetracycline, demeclocycline, meclocycline, doxycycline, lymecycline, methacycline, minocycline, oxytetracycline, rolitetracycline, and chlortetracycline. In embodiments, the tetracycline is doxycycline. In embodiments, the inducible suicide system is an HSV-TK system or an inducible caspase-9 system. In embodiments, the modified cells comprise a nucleic acid sequence encoding a suicide gene, such that when the modified cells are in the presence of a nucleoside analogue in a manner permitting expression of the suicide gene, to render the nucleoside analogue cytotoxic to the modified cells. In embodiments, the suicide gene is selected from the group consisting of thymidine kinase of herpes simplex virus, thymidine kinase of varicella zoster virus, and bacterial cytosine deaminase. In embodiments, the suicide gene is thymidine kinase of herpes simplex virus. In embodiments, the nucleoside analogue is selected from the group consisting of ganciclovir, acyclovir, buciclovir, famciclovir, penciclovir, valciclovir, trifluorothymidine, 1-[2-deoxy, 2-fluoro, beta-D-arabino furanosyl]-5-iodouracil, ara-A, araT 1-beta-D-arabinofuranoxyl thymine, 5-ethyl-2′-deoxyuridine, 5-iodo-5′-amino-2,5′-dideoxyuridine, idoxuridine, AZT, AIU, dideoxycytidine, and araC. In embodiments, the nucleoside analogue is ganciclovir.

In embodiments, expression of the one or more molecules is regulated by one or more promoters. In embodiments, the polynucleotide comprises a promoter comprising a binding site for a transcription modulator that modulates the expression and/or secretion of the one or more molecules in the cell. For example, the transcription modulator is or includes Hif1a, NFAT, FOXP3, and/or NFkB. For example, the one or more molecules comprise at least one cytokine associated with an oxygen-sensitive polypeptide domain, and the oxygen-sensitive polypeptide domain comprises HIF VHL binding domain.

In embodiments, the polynucleotide may integrate into the genome of the modified cell and descendants of the modified cell will also express the polynucleotide, resulting in a stably transfected modified cell. In embodiments, the modified cell may express the polynucleotide encoding the CAR but the polynucleotide does not integrate into the genome of the modified cell such that the modified cell expresses the transiently transfected polynucleotide for a finite period of time (e.g., several days), after which the polynucleotide is lost through cell division or other factors. For example, the polynucleotide is present in the modified cell in a recombinant DNA construct, in an mRNA, or in a viral vector, and/or the polynucleotide is an mRNA, which is not integrated into the genome of the modified cell.

Embodiments relate to a method or use of polynucleotide. The method or use includes: providing a viral particle (e.g., AAV, lentivirus or their variants) comprising a vector genome, the vector genome comprising the polynucleotide, wherein the polynucleotide is operably linked to an expression control element conferring transcription of the polynucleotide; and administering an amount of the viral particle to the subject such that the polynucleotide is expressed in the subject. In embodiments, the AAV preparation may include AAV vector particles, empty capsids and host cell impurities, thereby providing an AAV product substantially free of AAV empty capsids. More information of the administration and preparation of the viral particle may be found at the U.S. Pat. No. 9,840,719 and Milani et al., Sci. Transl. Med. 11, eaav7325 (2019) 22 May 2019, which are incorporated herein by reference.

In embodiments, the bioreactor may be inoculated at a cell density of approximately 0.5×10⁶ cells/mL with viability greater than 95%. When the cell density reaches approximately 1.0×10⁶cells/mL, the cells may be transfected with the PEI/DNA complexes (polyplexes) with a PEI to DNA ratio of 2:1. At the time of harvest, AAV from the cell culture in the bioreactor may be released using the Triton X-100 method. All solutions may be added directly to the bioreactor, and the lysate was centrifuged at 4000×g for 20 min. The supernatant may be stored at −80° C. for further processing. AAV may be further purified. For example, AAV samples (12.3 mL) may be purified by overlaying them on top of series of step gradients using 15, 25, 40 and 54% iodixanol concentrations containing 1, 5, 7 and 5 mL, respectively. The 15% iodixanol concentration also contains 1 M NaCl to avoid aggregation of AAV with other cellular proteins and negatively charged nuclear components. After the completion of centrifugation, 5 mL may be withdrawn from 2 mm below the 40/54 interface marked before starting the ultracentrifugation at 385,000×g for 1 h 45 min in Sorvall T-865 rotor in Sorvall Ultracentrifuge. The viral vectors may be then quantified. For example, vectors AAV infectivity may be determined by the gene transfer assay (GTA) using GFP as a reporter gene in all cases. AAV infectivity assay where sample may be diluted before addition to the cells to have the GFP positive cells in the range of 2-20% to assure that only single virus has entered the cell for GFP expression. The GFP-positive cells may be quantified by FACS using HEK293 cells in suspension. The AAV may be then administrated to a subject. For example, AAV may be diluted in 0.9% sterile NaCl saline solution (supplemented with 0.25% human serum albumin [HSA]) for infusion in patients and the final volume of infusion will be calculated based on the patient's weight as 3 mL/kg.

The term “expression or overexpression” refers to the transcription and/or translation of a particular nucleotide sequence into a precursor or mature protein, for example, driven by its promoter. “Overexpression” refers to the production of a gene product in transgenic organisms or cells that exceeds levels of production in normal or non-transformed organisms or cells. As defined herein, the term “expression” refers to expression or overexpression.

In embodiments, a T cell clone that expresses a TCR with a high affinity for the target antigen may be isolated. Tumor-infiltrating lymphocytes (TILs) or peripheral blood mononuclear cells (PBMCs) can be cultured in the presence of antigen-presenting cells (APCs) pulsed with a peptide representing an epitope known to elicit a dominant T cell response when presented in the context of a defined HLA allele. High-affinity clones may be then selected on the basis of MHC-peptide tetramer staining and/or the ability to recognize and lyse target cells pulsed with low titrated concentrations of cognate peptide antigen. After the clone has been selected, the TCRα and TCRβ chains or TCRγ and TCRδ chains are identified and isolated by molecular cloning. For example, for TCRα and TCRβ chains, the TCRα and TCRβ gene sequences are then used to generate an expression construct that ideally promotes stable, high-level expression of both TCR chains in human T cells. The transduction vehicle, for example, a gammaretrovirus or lentivirus, can then be generated and tested for functionality (antigen specificity and functional avidity) and used to produce a clinical lot of the vector. An aliquot of the final product can then be used to transduce the target T cell population (generally purified from patient PBMCs), which is expanded before infusion into the patient.

Various methods may be implemented to obtain genes encoding tumor-reactive TCR. More information is provided in Kershaw et al., Clin Transl Immunology. 2014 May; 3(5): e16. In embodiments, specific TCR can be derived from spontaneously occurring tumor-specific T cells in patients. Antigens included in this category include the melanocyte differentiation antigens MART-1 and gp100, as well as the MAGE antigens and NY-ESO-1, with expression in a broader range of cancers. TCRs specific for viral-associated malignancies can also be isolated, as long as viral proteins are expressed by transformed cells. Malignancies in this category include liver and cervical cancer, associated with hepatitis and papilloma viruses, and Epstein-Barr virus-associated malignancies. In embodiments, target antigens of the TCR may include CEA (e.g., for colorectal cancer), gp100, MART-1, p53 (e.g., for Melanoma), MAGE-A3 (e.g., Melanoma, esophageal and synovial sarcoma), NY-ESO-1 (e.g., for Melanoma and sarcoma as well as Multiple myelomas).

In embodiments, preparation and transfusion of tumor infiltrating lymphocytes (TIL) may be implemented by the following. For example, tumor tissue comes from surgical or biopsy specimens, may be obtained under aseptic conditions and transported to the cell culture chamber in ice box. Necrotic tissue and adipose tissue may be removed. The tumor tissue may be cut into small pieces of about 1-3 cubic millimeter. Collagenase, hyaluronidase and DNA enzyme may be added, and digested overnight at 4° C. Filtering with 0.2 um filter, cells may be separated and collected by lymphocyte separation fluid, 1500 rpm for 5 min. Expanding the cells with a culture medium comprising PHA, 2-mercaptoethanol and CD3 monoclonal antibody, a small dose of IL-2 (10-20 IU/ml) may be added to induce activation and proliferation. According to the growth situation, the cell density may be carefully detected and maintained within the range of 0.5-2*10{circumflex over ( )}6/ml under the condition of 37° C. and 5% CO2 for 7-14 days. TIL positive cells have the ability to kill homologous cancer cell may be screened out by co-culture. The positive cells may be amplified in a serum-free medium containing a high dose of IL2 (5000-6000 IU/ml) until greater than 1*10{circumflex over ( )}11 TILs may be obtained. To administer TILs, they may be first collected in saline using continuous-flow centrifugation and then filtered through a platelet-administration set into a volume of 200-300 mL containing 5% albumin and 450 000 IU of IL-2. The TILs may be infused into patients through a central venous catheter over a period of 30-60 minutes. In embodiments, TILs may be often infused in two to four separate bags; the infusions may be separated by several hours.

In embodiments, expression of the polynucleotide is regulated or modulated by a synthetic Notch receptor comprising, from N-terminal to C-terminal and in covalent linkage: a) an extracellular domain comprising an antibody (e.g., a single-chain Fv (scFv) or a nanobody) that specifically binds to an antigen; b) a Notch regulatory region (NRR) and c) an intracellular domain comprising a transcriptional activator comprising a DNA binding domain. In embodiments, the Notch regulatory region comprises a Lin 12-Notch repeat, a heterodimerization domain comprising an S2 proteolytic cleavage site and a transmembrane domain comprising an S3 proteolytic cleavage site. The intracellular domain is heterologous to the Notch regulatory region. In embodiments, the transcriptional activator replaces a naturally-occurring intracellular notch domain, and binding of the antibody to the antigen induces cleavage at the S2 and S3 proteolytic cleavage sites, thereby releasing the intracellular domain. The release of the intracellular domain causes the transcriptional activator to induce expression of the polynucleotide encoding one or more target proteins in the modified cell. In embodiments, the modified cell comprises a polynucleotide encoding the synthetic Notch receptor and a polynucleotide encoding a transcriptional control element that is responsive to the transcriptional activator and operably linked to the polynucleotide encoding one or more target proteins (e.g., CAR and scFv targeting M2).

“Suicide gene” is a nucleic acid coding for a product, wherein the product causes cell death by itself or in the presence of other compounds. A representative example of such a therapeutic nucleic acid (suicide gene) is one which codes for thymidine kinase of herpes simplex virus (HSV-TK). Additional examples are thymidine kinase of varicella zoster virus and the bacterial gene cytosine deaminase which can convert 5-fluorocytosine to the highly toxic compound 5-fluorouracil.

In embodiment, the modified cell comprises an inducible gene expression system that comprises or is a lac system, a tetracycline system, or a galactose system. In embodiment, the expression of the dominant negative form of the inhibitory immune checkpoint molecule is regulated by Hif1a, NFAT, FOXP3, and/or NFkB.

In embodiment, the modified cell comprises a polynucleotide encoding the binding molecule and a dominant negative form of the inhibitory immune checkpoint molecule or a receptor thereof, wherein expression of the dominant negative form of the inhibitory immune checkpoint molecule is regulated by an inducible gene expression system.

In embodiment, the modified cell comprises a polynucleotide encoding the dominant negative form of the inhibitory immune checkpoint molecule or the receptor thereof, wherein expression of the dominant negative form of the inhibitory immune checkpoint molecule is regulated by an inducible gene expression system. In embodiments, the modified cell comprises a sequence listed in Table 7.

In embodiment, the polynucleotide comprises a polynucleotide encoding a NFAT promoter operatively associated with a nucleotide sequence encoding the inhibitory immune checkpoint molecule or the receptor thereof.

Embodiments relate to a modified cell comprising a polynucleotide encoding a secretable antibody binding SIGLEC-15 and/or encoding a dominant negative form of CD44. The antibody binding SIGLEC-15 can be a scFv. Embodiments relate to a modified cell comprising a polynucleotide encoding a secretable scFv binding SIGLEC-15 and/or encoding a secretable scFv binding CD44. In embodiments, the scFv binding SIGLEC-15 or CD44 is attached to the membrane of the modified cell as shown in FIG. 1.

Embodiments relate to a fusion protein comprising a scFv binding SIGLEC-15, a linker, an extracellular domain, a transmembrane domain, and a cytoplasmic domain, wherein the transmembrane domain is selected from a group consist of a transmembrane domain of a receptor of IL15, IL2, IL7, IL6, IL12, IL18, IL21, IL23, IL 33, TNFα, TNFβ, IFNα, and IFNβ, and the cytoplasmic domain is selected from a group consist of a cytoplasmic domain of receptor of the receptor of IL15, IL2, IL7, IL6, IL12, IL18, IL21, IL23, IL 33, TNFα, TNFβ, IFNα, and IFNβ, and the extracellular domain is selected from a group consist of an extracellular domain of the receptor of IL15, IL2, IL7, IL6, IL12, IL18, IL21, IL23, IL 33, TNFα, TNFβ, IFNα, and IFNβ. Embodiments relate to a fusion protein comprising a scFv binding SIGLEC-15, a linker, a transmembrane domain, and a cytoplasmic domain, wherein the transmembrane domain is selected from a group consist of a transmembrane domain of a receptor of CD4, CD8, CD28, CD27, CD25, CD137, PD1 and PDL1, and the cytoplasmic domain is selected from a group consist of a cytoplasmic domain of receptor of the receptor of CD4, CD8, CD28, CD27, CD25, CD137, PD1 and PDL1. Embodiments relate to a nucleic acid sequence encoding the fusion protein.

In embodiments, the binding domain is a scFv. In embodiments, the link is a GS linker. In embodiments, the scFv binds to one of the SEQ ID NOs: 85-89, the extracellular domain is or comprises a SEQ ID NO: 36-42, the transmembrane domain is or comprises one of the SEQ ID NOs: 43-49, and/or the cytoplasmic domain is or comprises one of the SEQ ID NO: 50-56; or the scFv binds to one of the SEQ ID NOs: 85-89, the liner is or comprises a SEQ ID NO: 83 and 84, the transmembrane domain is or comprises one of the SEQ ID NOs: 59-63, and/or the cytoplasmic domain is or comprises one of the SEQ ID NO: 64-70.

Embodiments relate to a nucleic acid sequence encoding a dominant negative form of SIGLEC-15 receptor on T cells or the dominant negative form of SIGLEC-15 receptor and CAR or modified TCR, wherein the modified SIGLEC-15 receptor and the CAR/TCR are expressed as gene products that are separate polypeptides. Embodiments relate to a nucleic acid sequence encoding a dominant negative form of SIGLEC-15 receptor on T cells (e.g., CD44) or the dominant negative form of CD44 and CAR or modified TCR, wherein the modified CD44 and the CAR/TCR are expressed as gene products that are separate polypeptides. Dominant negative mutations have an altered gene product that acts antagonistically to the wild-type allele. These mutations usually result in an altered molecular function (often inactive) and are characterized by a dominant or semi-dominant phenotype.

In embodiments, the dominant negative form of CD44 comprises a substitution or deletion as compared to a wild-type CD44 intracellular domain. SIGLEC-15 is a member of the SIGLEC gene family and has a typical sialic acid-binding immunoglobulin-type lectin structure, which plays a very important role in osteoclast differentiation and bone remodeling [1-4]. Expression of SIGLEC-15 is typically restricted to myeloid cells and is up-regulated in many human cancers. SIGLEC-15 is an immunosuppressive molecule that acts primarily on the tumor microenvironment and has no correlation with the PD-L1/PD-1 pathway. CD44 may be a potential functional ligand for SIGLEC-15 and is involved in the activation of T cells. The dominant negative receptor is constructed, that is, the extracellular segment of CD44 is retained, and its transmembrane or intracellular segment is modified to bind to SIGLEC-15, but the inhibitory signal cannot be transmitted through its cytoplasmic domain such as to enhance the anti-tumor activity of CAR-T cells.

Embodiments relate to a modified cell comprises the fusion protein or the nucleic acid sequence. Embodiments relate to a modified cell comprises a dominant negative form of CD44. In embodiments, the dominant negative form of CD44 is or comprises one of the SEQ ID NOS: 94-96. In embodiments, the modified cell is a T cell, NK cell, or dendritic cells.

Embodiments relate to a pharmaceutical composition comprising the population of the modified cells. Embodiments relate to a method of cause T cell response in a subject in need thereof and/or treating a tumor of the subject, the method comprising administering an effective amount of the composition to the subject.

Embodiments relate to a method of causing T cell response, treating a subject having cancer, enhancing cellular treatment on a subject having cancer, or inhibiting growth of tumor cells, the method comprising: administering an effective amount of modified cells comprising a binding molecule to the subject; and administering an effective amount of an inhibitor of an inhibitory immune checkpoint molecule (e.g., molecules associated with SIGLEC-15 and CD44 pathway) or a receptor thereof in response to a predetermined condition. In embodiments, the method further comprises intruding into cells with a plurality of nucleic acid sequences encoding the binding molecule to obtain the modified cells. In embodiments, the method further comprises monitoring cytokine releases of the subject in response to the CAR infusion; and wherein the administering the effective amount of the inhibitor of the inhibitory immune checkpoint molecule or the receptor thereof in response to the predetermined condition comprises administering the effective amount of the inhibitor of the inhibitory immune checkpoint molecule or the receptor thereof in response to determination that a level of one or more cytokines increases and/or reaches a peak level. In embodiments, the method further comprises terminating the administering of the inhibitor in response to determination that the level drops to a base level. In embodiments, the cytokine may include IL6 and/or IFNγ, as shown in FIG. 11.

In embodiments, the modified cell comprises a CAR. In embodiments, the CAR comprises an extracellular domain, a transmembrane domain, and an intracellular domain (e.g., 41-BB and CD3 zeta domain), the extracellular domain binds an antigen. In embodiments, the intracellular domain comprises a costimulatory signaling region that comprises an intracellular domain of a costimulatory molecule selected from the group consisting of CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and any combination thereof. In embodiments, the antigen is Epidermal growth factor receptor (EGFR), Variant III of the epidermal growth factor receptor (EGFRvIII), Human epidermal growth factor receptor 2 (HER2), Mesothelin (MSLN), Prostate-specific membrane antigen (PSMA), Carcinoembryonic antigen (CEA), Disialoganglioside 2 (GD2), Interleukin-13Ra2 (IL13Rα2), Glypican-3 (GPC3), Carbonic anhydrase IX (CAIX), L1 cell adhesion molecule (L1-CAM), Cancer antigen 125 (CA125), Cluster of differentiation 133 (CD133), Fibroblast activation protein (FAP), Cancer/testis antigen 1B (CTAG1B), Mucin 1 (MUC1), Folate receptor-α (FR-α), CD19, FZD10, TSHR, PRLR, Muc 17, GUCY2C, CD207, CD3, CD5, B-Cell Maturation Antigen (BCMA), or CD4.

TABLE 2 SEQ ID NO: Identity 1 SP 2 Hinge & transmembrane domain 3 Co-stimulatory region 4 CD3-zeta 5 scFV Humanized CD19 6 scFV CD19 7 scFv FZD10 8 scFv TSHR 9 scFv PRLR 10 scFv Muc 17 11 scFv GUCY2C 12 scFv CD207 13 Prolactin (ligand) 14 scFv CD3 15 scFv CD4 16 scFv CD4-2 17 scFv CD5 18 WTCD3zeta 19 WTCD3zeta-BCMACAR full length 20 BCMACAR 21 MUC1CAR 22 m19CAR-IRES-MUC1CAR 23 hCD19CAR-IRES-MUC1CAR 24 hCD22CAR-IRES-MUC1CAR 25 BCMACAR-IRES-MUC1CAR 26 mCD19CAR-2A-MUC1CAR 27 hCD19CAR-2A-MUC1CAR 28 hCD22CAR-2A-MUC1CAR 29 BCMA-2A-MUC1CAR 30 Tumor associated MUC1 scFv 1 31 Tumor associated MUC1 scFv-1 VH 32 Tumor associated MUC1 scFv-1 VL 33 Tumor associated MUC1 scFv 2 34 Tumor associated MUC1 scFv2 VH 35 Tumor associated MUC1 scFv2 VL 36 ED IL2 receptor 37 ED IL6 receptor 38 ED IL7 receptor 39 ED IL12 receptor 40 ED IL15 receptor 41 ED IL21 receptor 42 ED IL23 receptor 43 TM IL2 receptor 44 TM IL6 receptor 45 TM IL7 receptor 46 TM IL12 receptor 47 TM IL15 receptor 48 TM IL21 receptor 49 TM IL23 receptor 50 CD IL2 receptor 51 CD IL6 receptor 52 CD IL7 receptor 53 CD IL12 receptor 54 CD IL15 receptor 55 CD IL21 receptor 56 CD IL23 receptor 57 TM CD4 58 TM CD8 59 TM CD27 60 TM CD28 61 TM CD137 62 TM PD1 63 TM PDL1 64 CD CD4 65 CD CD8 66 CD CD27 67 CD CD28 68 CD CD137 69 CD PD1 70 CD PDL1 71 IL2 72 IL6 73 IL7 74 IL12 75 IL15 76 IL21 77 IL23 78 IL33 79 TNFα 80 TNFβ 81 Hif VHL-interaction domain: Hif amino acid 344-417 82 Hif amino acid 380-603 83 GS linker sequence 84 EA linker sequence 85 SIGLEC-15 antigen 1 86 SIGLEC-15 antigen 2 87 SIGLEC-15 antigen 3 88 SIGLEC-15 antigen 4 89 SIGLEC-15 antigen 5 90 SIGLEC-15 antigen 6 91 SIGLEC-15 antigen 7 92 SIGLEC-15 antigen 8 93 CD44 VVT aa 94 Truncated CD44 aa 95 CD44 + CD8α aa 96 CD44 point mutant aa 97 h-Notch transmembrane 98 m-Notch transmembrane 99 OX40 extracellular 100 CD40 extracelluar 101 41-BB extracellular 102 Her2 extracellular 103 GITR extracellular 104 CD28 extracellular 105 ICOS extracellular 106 EGFR extracellular 107 CD27 extracellular 108 OPA1 109 MKN1 110 MKN2 111 DNM1L 112 RUNX3 113 EOMES 114 SIGLEC-15-P2A-EGFP 115 Anti-SIGLEC-15 scFv1 116 Anti-SIGLEC-15 scFv2 117 DNM1L 118 mCD19-P2A-DNM1L 119 P2A-DNM1L Note: EM: Extracellular Domain; TM: Transmembrane Domain; CD: Cytoplasmic Domain

Embodiments relate to a fusion protein comprising an extracellular domain, a transmembrane domain, and a cytoplasmic domain, the extracellular domain being or comprising the extracellular domain of at least one of OX40, CD40, 41-BB, GITR, ICOS, CD28, CD27, HER, or EGFR, the transmembrane domain being or comprising the transmembrane domain of an inducible molecule, and the cytoplasmic domain being or comprising at least one of OPA1, MKN1, MKN2, DNM1L, runx3, EOMES, TCF1, LEF1, Runx1, STAT3, TRAF6, ID3.

Embodiments relate to a fusion protein comprising an extracellular domain, a transmembrane domain, and a cytoplasmic domain, the transmembrane domain being or comprising the transmembrane domain of Notch. More information about Notch and its components may be found in U.S. Pat. No. 9,670,281, which incorporated herein by reference.

In embodiments, the extracellular domain comprises an extracellular domain of a receptor, an antibody, or a ligand. In embodiments, the receptor is a costimulatory molecule. In embodiments, the receptor is OX40, CD40, 41-BB, GITR, ICOS, CD28, CD27, HER, or EGFR. In embodiments, the ligand corresponds to the receptor. In embodiments, the receptor is a cytokine receptor, and the ligand is a cytokine. In embodiments, the antibody is an scFv targeting a tumor antigen. In embodiments, the cytoplasmic domain comprises a mitochondrial protein or a transcription factor. In embodiments, the cytoplasmic domain comprises at least one of OPA1, MKN1, MKN2, DNM1L, runx3, EOMES, TCF1, LEF1, Runx1, STAT3, TRAF6, and ID3. In embodiments, the cytoplasmic domain comprises at least one of a protein listed in Table 3.

In embodiments, the extracellular domain corresponds to an agent, and the transmembrane domain of Notch comprises a TACE cleavage site and a γ-secretase cleavage site such that binding of an extracellular domain to the agent induces cleavage at the TACE cleavage site and the γ-secretase cleavage site, thereby releasing the cytoplasmic domain. In embodiments, the agent is the receptor, and the extracellular domain comprises the ligand of the receptor. In embodiments, the extracellular domain comprises an extracellular domain of the receptor, and the agent is a ligand corresponding to the receptor. In embodiments, the agent is the tumor antigen, and the extracellular domain comprises an antibody against the tumor antigen.

In embodiments, the extracellular domain comprises at least one of SEQ ID NO: 38-46, the transmembrane domain comprises at least one of SEQ ID NO: 36 and 37, and the cytoplasmic domain comprises at least one of SEQ ID NO: 47-52.

Embodiments relate to a nucleic acid sequence encoding the fusion protein. Embodiments relate to a modified cell comprises a fusion protein described herein, for example one of embodiments 1-16 or the nucleic acid encoding a fusion protein described herein (one of embodiments 1-16). Embodiments relate to a pharmaceutical composition comprising the population of the modified cells. Embodiments relate to a method of cause T cell response in a subject in need thereof and/or treating a tumor of the subject, the method comprising administering an effective amount of the composition.

In embodiments, the modified cell comprises a CAR. In embodiments, the CAR comprises an extracellular domain, a transmembrane domain, and an intracellular domain, and the extracellular domain binds an antigen. In embodiments, the pharmaceutical composition, the modified cell, or the method of promoting a T cell response described herein include the intracellular domain comprises a costimulatory signaling region that comprises an intracellular domain of a costimulatory molecule selected from the group consisting of CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and any combination thereof. In embodiments, the antigen is Epidermal growth factor receptor (EGFR), Variant III of the epidermal growth factor receptor (EGFRvIII), Human epidermal growth factor receptor 2 (HER2), Mesothelin (MSLN), Prostate-specific membrane antigen (PSMA), Carcinoembryonic antigen (CEA), Disialoganglioside 2 (GD2), Interleukin-13Ra2 (IL13Rα2), Glypican-3 (GPC3), Carbonic anhydrase IX (CAIX), L1 cell adhesion molecule (L1-CAM), Cancer antigen 125 (CA125), Cluster of differentiation 133 (CD133), Fibroblast activation protein (FAP), Cancer/testis antigen 1B (CTAG1B), Mucin 1 (MUC1), Folate receptor-α (FR-α), CD19, FZD10, TSHR, PRLR, Muc 17, GUCY2C, CD207, CD3, CD5, B-Cell Maturation Antigen (BCMA), or CD4. In embodiments, the cell is a T cell, macrophage, dendritic cell, or NK cell.

Embodiments relate to a fusion protein that can be activated by agonist antibodies to perform the specific desired functions. In an overexpressed fusion protein, the extracellular domain comprise a protein sequence that can be bound by an agonist antibody (that is, an antibody that binds the extracellular domain of the fusion protein), and the intracellular is the intracellular domain of another transmembrane protein. When using the agonist antibody, the extracellular segment is cross-linked by the agonist antibody, and the intracellular segment of the fusion protein forms a cluster that activates the downstream pathway. Examples of the fusion protein and the agonist antibody are shown in FIG. 12. An “agonist antibody,” as used herein, is an antibody which activates a biological activity of the antigen it binds. In embodiments, during agonist antibody activation, partial dissociation of antibodies allows the antigen-binding fragment (Fab) arms of a single antibody to interact with more than two receptors in a dynamic fashion, resulting in the recruitment of multiple receptor monomers into a receptor oligomer where signaling activation can be triggered. In embodiments, antigen-presenting cells (APCs) acts as a scaffold to crosslink agonist antibody bound to a receptor (e.g., co stimulatory receptor), leading to receptor supercluster formation and increased agonist signaling. Some proteins (such as the proteins of the CD28 family and the TNF receptor family) have an agonist antibody for tumor therapy. More information about agonist antibodies and co-stimulatory molecules may be found at Nature Reviews Drug Discovery volume 17, pages 509-527 (2018), which is incorporated herein by reference in its entirety.

TABLE 3 Group 1 Group 1 Group 1 Group 1 Group 2 Group 3 Group 4 LEF1 CXCR3 FRC T-bet PI3K ezh2 PDCD1 Gfi1 Flt3 FAO GATA3 Akt kiaa1324 PDL1 δEF1/ZEB Kit Hif-1a Blimp-1 mTOR fn1 PDL2 Ikaros IL-7R Hif-2a Myc Wnt ITGA9 CTLA4 KLF2 IL-4R Vhl HIF1 Notch1 SMDPD3A LRBA HEB IL-9R PHD1 c-Myb Notch2 ITPRIPL2 LAG3 MAZR PDGFRb PHD2 members of the Activin PRSS23 Tim3 E2A/HEB family Tox gc (Il2rg PHD3 members of the BMP CLECL1 BILA Ikaros family PU.1 IL-2Ra FIH1 Ets-family TGFb TBX21 CD160 (CD25) transcription factor PU.1 HES1,2,3 IL-2Rb TET2 Runx1 IL7 IKZF2 2B4 Sox4 PDGFRa TET1 GATA-2 IL7Ra EOMES Foxp3 E12, Pdgfa TET3 TCF-1 (Tcf7) IL12 PRDM1 ccr4 E2-2 Pdgfb DNMT3 RBPSuh IL15 BTLA PVRIG E47, Pdgfc Id3 IL6 CD244 CD16B MEF2 Pdgfd Id2 IL2 KL6 SIVA1 Nur77/Nor1 TGF-b1 Runx1 MAPK JUNB CD33 Ncoa4 TGF-b2 Runx3 AMPK FOSB LAGLS9 Basp2 TGF-b3 EBF NF-Kb FAM13A CD122 Pitx1 BMP2 Pax5 NF-AT BATF3 IDO1 Prdm16 BMP4 FOG-1 AP-1 KLRC1 IDO2 Ndn BMP7 GATA-3 PI-3 KLRC2 CD45 kinase, Akt/PKB, and Ras/MAP Irf6 ActivinbetaA GATA-2 CCR7 ZNF704 CVPLBL Dach1 ALK-5 GATA-1 STAT1 CTHRC1 TNFAIP8L2 Nr4a2 BMPR-1A/B Groucho/TLE/ STAT2 FAXC DNMT3A Grg family proteins or Sin3A Hoxa5 BMPR-II Helios STAT3 EGR1 CEACAM-1 Hoxb5 TLR NFAT STAT4 RBM47 RUNX3 FoxN1 MyD88 Bcl-6 STAT5 ENTPD1 LEXM Gli1,2,3 SHH BCL-6b (BAZF) glut1 SUV39H1 PILRA Smoothen HIF1 AKAP5 PTNNS1L3 Ptch1 c-Myc β-catenin Fegr3a Fu IRF4 PI3K Nat8 Su(fu) NF-kb GLUL ccl9 Wnt1,3,4,5b,10b ThPOK FAXC HCK Smad Oct3/4 TREM2 CXCL10 Nanog LNGM CCL6 CXCL9 Sox2 NUDT16 CD36 INFa,b KLF4 IGF1 CXCR3 FOXO1 CTSS CCL5 TSC1 GZMC CCR5 OxPhos BATF CCR7 Zbtb32 CXCL2 SOCS1 E2F2 TNFAIP8L 3 SOCS2 SMART IL-1b SOCS3 NCOR IL-1a SOCS4 TRPV1 SOCS5 TRPV2 SOCS6 TRPV3 SOCS7 TRPV4 CIS Rgs1 PLSCR1 ITGB1 ITGB2 C3AR1 ITGA3 ITGA5 ITGAL CARD11 CD83

The present disclosure is further described by reference to the following exemplary embodiments and examples. These exemplary embodiments and examples are provided for purposes of illustration only and are not intended to be limiting unless otherwise specified. Thus, the present disclosure should in no way be construed as being limited to the following exemplary embodiments and examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

EXEMPLARY EMBODIMENTS

The following are exemplary embodiments:

-   1. A fusion protein comprising an extracellular domain, a     transmembrane domain, and a cytoplasmic domain, the extracellular     domain being or comprising an extracellular domain of at least one     of OX40, CD40, 41-BB, GITR, ICOS, CD28, CD27, HER, or EGFR, the     transmembrane domain being or comprising a transmembrane domain of     an inducible molecule, and the cytoplasmic domain being or     comprising at least one of OPA1, MKN1, MKN2, DNM1L, runx3, EOMES,     TCF1, LEF1, Runx1, STAT3, TRAF6, or ID3. -   2. A fusion protein comprising an extracellular domain, a     transmembrane domain, and a cytoplasmic domain, the transmembrane     domain being or comprising the transmembrane domain of Notch. -   3. The fusion protein of embodiment 2, wherein the extracellular     domain comprises an extracellular domain of a receptor, an antibody,     or a ligand. -   4. The fusion protein of embodiment 3, wherein the receptor is a     costimulatory molecule. -   5. The fusion protein of embodiment 3, wherein the receptor is OX40,     CD40, 41-BB, GITR, ICOS, CD28, CD27, HER, or EGFR. -   6. The fusion protein of embodiment 3, wherein the ligand     corresponds to a receptor. -   7. The fusion protein of embodiment 3, wherein the receptor is a     cytokine receptor, and the ligand is a cytokine. -   8. The fusion protein of embodiment 3, wherein the antibody is a     scFv that targets a tumor antigen. -   9. The fusion protein of one of embodiments 2-8, wherein the     cytoplasmic domain comprises a mitochondrial protein or a     transcription factor. -   10. The fusion protein of embodiment 9, wherein the cytoplasmic     domain comprises at least one of OPA1, MKN1, MKN2, DNM1L, runx3,     EOMES, TCF1, LEF1, Runx1, STAT3, TRAF6, or ID3. -   11. The fusion protein of embodiment 9, wherein the cytoplasmic     domain comprises at least one of a protein listed in Table 3. -   12. The fusion protein of one of embodiments 2-11, wherein the     extracellular domain corresponds, to, interacts or associates with,     and/or binds an agent, and the transmembrane domain of Notch     comprises a TACE cleavage site and a y-secretase cleavage site such     that binding of an extracellular domain to the agent induces     cleavage at the TACE cleavage site and the y-secretase cleavage     site, thereby releasing the cytoplasmic domain. -   13. The fusion protein of embodiment 12, wherein the agent is a     receptor, and the extracellular domain comprises a ligand of the     receptor. -   14. The fusion protein of embodiment 12, wherein the extracellular     domain comprises an extracellular domain of a receptor, and the     agent is a ligand of the receptor. -   15. The fusion protein of embodiment 12, wherein the agent is a     tumor antigen, and the extracellular domain comprises an antibody     against the tumor antigen. -   16. The fusion protein of embodiment 12, wherein the extracellular     domain comprises an amino acid sequence of at least one of SEQ ID     NO: 99-106, the transmembrane domain comprises an amino acid     sequence of at least one of SEQ ID NO: 97 and 98, and the     cytoplasmic domain comprises an amino acid sequence of at least one     of SEQ ID NO: 108-113. -   17. A nucleic acid encoding the fusion protein of one of embodiments     1-16. -   18 A modified cell comprising the fusion protein of one of     embodiments 1-16 or the nucleic acid sequence of embodiment 17. -   19. A pharmaceutical composition comprising a population of the     modified cells of embodiment 18. -   20. A method of causing or promoting a T cell response in a subject     in need thereof and/or treating a tumor of the subject, the method     comprising administering an effective amount of the composition of     embodiment 19 to the subject. -   21. The pharmaceutical composition, the modified cell, and the     method of one of embodiments 17-20, wherein the modified cell     comprises a CAR. -   22. The pharmaceutical composition, the modified cell, and the     method of one of embodiments 17-20, wherein the CAR comprises an     extracellular domain, a transmembrane domain, and an intracellular     domain, and the extracellular domain binds an antigen. -   23. The pharmaceutical composition, the modified cell, and the     method of embodiment 23, wherein the intracellular domain comprises     a costimulatory signaling region that comprises an intracellular     domain of a costimulatory molecule selected from the group     consisting of CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS,     lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT,     NKG2C, B7-H3, and a combination thereof. -   24. The pharmaceutical composition, the modified cell, and the     method of embodiment 23, wherein the antigen is Epidermal growth     factor receptor (EGFR), Variant III of the epidermal growth factor     receptor (EGFRvIII), Human epidermal growth factor receptor 2     (HER2), Mesothelin (MSLN), Prostate-specific membrane antigen     (PSMA), Carcinoembryonic antigen (CEA), Disialoganglioside 2 (GD2),     Interleukin-13Ra2 (IL13Rα2), Glypican-3 (GPC3), Carbonic anhydrase     IX (CAIX), L1 cell adhesion molecule (L1-CAM), Cancer antigen 125     (CA125), Cluster of differentiation 133 (CD133), Fibroblast     activation protein (FAP), Cancer/testis antigen 1B (CTAG1B), Mucin 1     (MUC1), Folate receptor-α (FR-α), CD19, FZD10, TSHR, PRLR, Muc 17,     GUCY2C, CD207, CD3, CD5, B-Cell Maturation Antigen (BCMA), or CD4. -   25. The pharmaceutical composition, the modified cell, and the     method of embodiments 18-24, wherein the cell is a T cell,     macrophage, dendritic cell, or NK cell. -   26. A fusion protein comprising an scFv binding SIGLEC-15, a linker,     an extracellular domain, a transmembrane domain, and a cytoplasmic     domain, wherein the transmembrane domain is selected from a group     consisting of a transmembrane domain of a receptor of IL15, IL2,     IL7, IL6, IL12, IL18, IL21, IL23, IL 33, TNFα, TNFβ, IFNα, and IFNβ,     and the cytoplasmic domain is selected from a group consisting of a     cytoplasmic domain of receptor of the receptor of IL15, IL2, IL7,     IL6, IL12, IL18, IL21, IL23, IL 33, TNFα, TNFβ, IFNα, and IFNβ, and     the extracellular domain is selected from a group consisting of an     extracellular domain of the receptor of IL15, IL2, IL7, IL6, IL12,     IL18, IL21, IL23, IL 33, TNFα, TNFβ, IFNα, and IFNβ. -   27. A fusion protein comprising an scFv binding SIGLEC-15, a linker,     a transmembrane domain, and a cytoplasmic domain, wherein the     transmembrane domain is selected from a group consisting of a     transmembrane domain of a receptor of CD4, CD8, CD28, CD27, CD25,     CD137, PD1, and PDL1, and the cytoplasmic domain is selected from a     group consisting of a cytoplasmic domain of receptor of the receptor     of CD4, CD8, CD28, CD27, CD25, CD137, PD1, and PDL1. -   28. The fusion protein of one of embodiments 26 and 27, wherein the     binding domain is an scFv. -   29. The fusion protein of one of embodiments 26-28, wherein the link     is a GS linker. -   30. The fusion protein of one of embodiments 26-28, wherein the scFv     binds one of the amino acid sequences of SEQ ID NOs: 85-89, the     extracellular domain is or comprises one of amino acid sequences of     SEQ ID NO: 36-42, the transmembrane domain is or comprises one of     the amino acid sequences of SEQ ID NOs: 43-49, and/or the     cytoplasmic domain is or comprises one of the amino acid sequences     of SEQ ID NO: 50-56; or the scFv binds to one of the amino acid     sequences of SEQ ID NOs: 85-89, the linker is or comprises SEQ ID     NO: 83 or 84, the transmembrane domain is or comprises one of amino     acid sequences of SEQ ID NOs: 59-63, and/or the cytoplasmic domain     is or comprises one of the amino acid sequences of SEQ ID NOs:     64-70. -   31. A nucleic acid sequence encoding the fusion protein of one of     embodiments 26-30. -   32. A nucleic acid sequence encoding a dominant negative form of     SIGLEC-15 receptor on T cells or the dominant negative form of     SIGLEC-15 receptor and CAR or modified TCR, wherein the modified     SIGLEC-15 receptor and the CAR/TCR are expressed as gene products     that are separate polypeptides. -   33. A nucleic acid sequence encoding a dominant negative form of     SIGLEC-15 receptor on T cells (e.g., CD44) or the dominant negative     form of CD44 and CAR or modified TCR, wherein the modified CD44 and     the CAR/TCR are expressed as gene products that are separate     polypeptides. -   34. The nucleic acid sequence of embodiment 33, wherein the dominant     negative form of CD44 comprises a substitution or deletion in its     intracellular domain as compared to a wild-type CD44. -   35 A modified cell comprising the fusion protein of one of     embodiments 1-6 or the nucleic acid sequence encoding one of the     fusion proteins of embodiments 9-10. -   36. A modified cell comprising a dominant negative form of CD44. -   37. The modified cell of embodiment 35 or 36, wherein the dominant     negative form of CD44 is or comprises one of the amino acid sequence     of SEQ ID NOs: 94-96. -   38. The modified cell of one of embodiments 35-37, wherein the     modified cell is a T cell, NK cell, or dendritic cells. -   39. A pharmaceutical composition comprising the population of the     modified cells of embodiment 38. -   40. A method of causing or promoting a T cell response in a subject     in need thereof and/or treating a tumor of the subject, the method     comprising administering an effective amount of the composition of     embodiment 39 to the subject. -   41. The pharmaceutical composition, the modified cell, and the     method of one of embodiments 35-40, wherein the modified cell     comprises a CAR. -   42. The pharmaceutical composition, the modified cell, and the     method of embodiment 41, wherein the CAR comprises an extracellular     domain, a transmembrane domain, and an intracellular domain (e.g.,     41-BB and CD3 zeta domain), the extracellular domain binds an     antigen. -   43. The pharmaceutical composition, the modified cell, and the     method of embodiment 41 or 42, wherein the intracellular domain     comprises a costimulatory signaling region that comprises an     intracellular domain of a costimulatory molecule selected from the     group consisting of CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS,     lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT,     NKG2C, B7-H3, and a combination thereof. -   44. The pharmaceutical composition, the modified cell, and the     method of embodiment 41 or 42, wherein the antigen is Epidermal     growth factor receptor (EGFR), Variant III of the epidermal growth     factor receptor (EGFRvIII), Human epidermal growth factor receptor 2     (HER2), Mesothelin (MSLN), Prostate-specific membrane antigen     (PSMA), Carcinoembryonic antigen (CEA), Disialoganglioside 2 (GD2),     Interleukin-13Ra2 (IL13Rα2), Glypican-3 (GPC3), Carbonic anhydrase     IX (CAIX), L1 cell adhesion molecule (L1-CAM), Cancer antigen 125     (CA125), Cluster of differentiation 133 (CD133), Fibroblast     activation protein (FAP), Cancer/testis antigen 1B (CTAG1B), Mucin 1     (MUC1), Folate receptor-α (FR-α), CD19, FZD10, TSHR, PRLR, Muc 17,     GUCY2C, CD207, CD3, CD5, B-Cell Maturation Antigen (BCMA), or CD4. -   45. A recombinant antigen-binding protein or fragment thereof     comprising the amino acid sequence of SEQ ID NO: 115 or 116. -   46. The recombinant antigen-binding protein of embodiment 45,     wherein said protein is an antibody. -   47. The recombinant antigen-binding protein of embodiment 46,     wherein the antibody is a human antibody. -   48. The recombinant antigen-binding protein of embodiment 46,     wherein said antibody or antigen-binding fragment thereof is intact     Ig, Fab, F(ab′)₂, Fv, or scFv. -   49. The antigen-binding protein of embodiment 45, wherein said     antigen-binding protein is a SIGLEC-15 agonist. -   50. The antigen-binding protein of embodiment 45, wherein said     antigen-binding protein is a SIGLEC-15 antagonist. -   51. The antigen-binding protein of embodiment 45, wherein said     antigen-binding protein is a chimeric antigen receptor (CAR). -   52. A nucleic acid encoding an antigen-binding protein of embodiment     45 or at least one of the amino acid sequences of SEQ ID NOS: 115,     116, 94-96, and 100. -   53. A vector comprising the nucleic acid of embodiment 52. -   54. A modified cell comprising an antigen-binding protein of     embodiment 45. -   55. A modified cell comprising a nucleic acid of embodiment 52. -   56. A modified cell comprising a vector of embodiment 53. -   57. An antigen-binding protein of embodiment 45 conjugated to a     therapeutic agent. -   58. The antigen-binding protein of embodiment 57, wherein said     therapeutic agent is a drug, toxin, radioisotope, protein, or     peptide. -   59. A pharmaceutical composition comprising an antigen-binding     protein of embodiment 45. -   60. A pharmaceutical composition comprising a nucleic acid of     embodiment 52. -   61. A pharmaceutical composition comprising a vector of embodiment     53. -   62. A pharmaceutical composition comprising a cell that expresses an     antigen-binding protein of embodiment 45. -   63. A method of increasing a T cell response in a subject comprising     administering to the subject a therapeutically effective amount of     an antigen-binding protein or an antigen-binding fragment thereof of     embodiment 45 or a nucleic acid that encodes the antigen-binding     protein or an antigen-binding fragment thereof of embodiment 45. -   64. The method of embodiment 63, wherein the antigen-binding protein     or antigen-binding fragment thereof inhibits, reduces, modulates or     abolishes signal transduction mediated by SIGLEC-15. -   65. A method for treatment of a subject having a SIGLEC-15 positive     disease, comprising administering to the subject a therapeutically     effective amount of an antigen-binding protein or an antigen-binding     fragment thereof of embodiment 45 or a nucleic acid that encodes the     antigen-binding protein or an antigen-binding fragment thereof of     embodiment 45. -   66. The method of embodiment 65, wherein said antigen-binding     protein or antigen-binding fragment thereof is a conjugate having a     cytotoxic moiety linked thereto. -   67. The method of embodiment 65, wherein the SIGLEC-15 positive     disease is cancer. -   68. A vector comprising a nucleic acid encoding a recombinant     anti-SIGLEC-15 antigen-binding protein of embodiment 45 and a     nucleic acid encoding a chimeric antigen receptor, wherein said     recombinant anti-SIGLEC-15 antigen-binding protein is not identical     to said chimeric antigen receptor. -   69. A modified cell comprising the vector of embodiment 68. -   68. A modified cell comprising a nucleic acid encoding a recombinant     anti-SIGLEC-15 antigen-binding protein of embodiment 45 and a     nucleic acid encoding a chimeric antigen receptor, wherein said     recombinant anti-SIGLEC-15 antigen-binding protein is not identical     to said chimeric antigen receptor. -   70. A modified cell comprising a recombinant SIGLEC-15     antigen-binding protein of embodiment 45 and a chimeric antigen     receptor, wherein said recombinant anti-SIGLEC-15 antigen-binding     protein is not identical to said chimeric antigen receptor. -   71. A vector or a cell comprising a vector that encodes a SIGLEC-15     antigen-binding protein of embodiment 45, wherein the recombinant     anti-SIGLEC-15 antigen-binding protein is an antibody. -   72. A vector or a cell of embodiment 71, wherein the recombinant     SIGLEC-15 antigen-binding protein is a human antibody. -   73. A vector or a cell of embodiment 71, wherein the recombinant     SIGLEC-15 antigen-binding protein is an intact Ig, Fab, F(ab′)2, Fv,     or scFv. -   74. A vector or cell of embodiment 71, wherein the recombinant     SIGLEC-15 antigen-binding protein is a SIGLEC-15 agonist. -   75. A vector or cell of embodiment 71, wherein the recombinant     SIGLEC-15 antigen-binding protein is a SIGLEC-15 antagonist. -   76. A vector or cell of embodiment 71, wherein the recombinant     anti-SIGLEC-15 antigen-binding protein is a secretable protein. -   77. A vector or cell of embodiment 71, wherein the chimeric antigen     receptor specifically binds to CD-19. -   78. A vector or cell of embodiment 71, wherein the chimeric antigen     receptor can be inserted in a human T cell membrane. -   79. The modified cell of any suitable preceding embodiments, wherein     the modified cell is a T cell, NK cell, and/or DC. -   80. A pharmaceutical composition comprising a vector or a cell of     any of embodiments 70-79. -   81. The pharmaceutical composition of embodiment 80 further     comprising a pharmaceutically acceptable carrier. -   82. A method of increasing a T cell response in a subject comprising     administering to the subject a therapeutically effective amount of a     vector or cell of any of embodiments 70-79, wherein the recombinant     SIGLEC-15 antigen-binding protein is a SIGLEC-15 antagonist. -   83. A method of increasing a T cell response in a subject comprising     administering to the subject a therapeutically effective amount of a     pharmaceutical composition comprising a vector or a cell of any of     embodiments 70-79, wherein the recombinant SIGLEC-15 antigen-binding     protein is a SIGLEC-15 antagonist. -   84. The method of embodiment 83, wherein the recombinant SIGLEC-15     antigen-binding protein inhibits, reduces, modulates or abolishes     signal transduction mediated by SIGLEC-15. -   85. A method of decreasing a T cell response in a subject comprising     administering to the subject a therapeutically effective amount of a     vector or cell of any of embodiments 70-79, or a pharmaceutical     composition of embodiment 80 or 81, wherein the recombinant     SIGLEC-15 antigen-binding protein is a SIGLEC-15 agonist. -   86. A method of decreasing a T cell response in a subject comprising     administering to the subject a therapeutically effective amount of a     vector or cell of any of embodiments 70-79, wherein the recombinant     SIGLEC-15 antigen-binding protein is a SIGLEC-15 agonist. -   87. A method of decreasing a T cell response in a subject comprising     administering to the subject a therapeutically effective amount of a     pharmaceutical composition of embodiment 80, wherein the recombinant     SIGLEC-15 antigen-binding protein is a SIGLEC-15 agonist. -   88. A method for treatment of a subject having a PD1-positive     disease, comprising administering to the subject a therapeutically     effective amount of a vector the encodes the or cell of any one of     any of embodiments 70-79, or a pharmaceutical composition of     embodiment 80 or 81. -   89. A method for treatment of a subject having a SIGLEC-15-positive     disease, comprising transducing at least one T cell of the subject     with a nucleic acid encoding a recombinant anti-SIGLEC-15     antigen-binding protein and a nucleic acid encoding a chimeric     antigen receptor, wherein said recombinant anti-SIGLEC-15     antigen-binding protein is not identical to said chimeric antigen     receptor. -   90. The method of embodiment 89, wherein the chimeric antigen     receptor does not specifically bind to SIGLEC-15. -   91. The method of embodiment 89, wherein the SIGLEC-15 positive     disease is cancer. -   92. A vector or a cell comprising a vector that encodes an     antigen-binding protein of any of embodiments 70-79, wherein at     least one of the anti-SIGLEC-15 antigen-binding protein and chimeric     antigen receptor is conjugated to a therapeutic agent. -   93. The vector or the cell of embodiment 92, wherein said     therapeutic agent is a drug, toxin, radioisotope, protein, or     peptide. -   94. The modified cell or the method of any suitable preceding     embodiments, wherein the modified cell is a T cell, NK cell, or     dendritic cell. -   95. A modified cell comprising a polynucleotide encoding a binding     molecule, and a polynucleotide encoding an anti-SIGLEC-15     antigen-binding. -   96. The modified cell of embodiment 95, wherein the anti-SIGLEC-15     antigen-binding protein is a secretable protein. -   97. The modified cell of embodiment 95, wherein the anti-SIGLEC-15     antigen-binding protein is a secretable scFv. -   98. The modified cell of embodiment 95, wherein the modified cell     comprises a dominant negative form of PD-1. -   99. The modified cell of any one of the preceding embodiments,     wherein the cell or modified cell is a T cell derived from a healthy     donor or a subject having cancer. -   100. The modified cell of any of preceding embodiments, wherein the     modified T cell comprises a dominant negative form of a receptor     associated with CD44. -   101. The modified cell of embodiment 100, wherein the modified CD44     lacks a functional CD44 intracellular domain for CD44 signal     transduction, interferes with a pathway between CD44 of a human T     cell of the human cells and SIGLEC-15 of a certain cell, comprises     or is a CD44 extracellular domain or a CD44 transmembrane domain or     a combination thereof, comprises a modified CD44 intracellular     domain comprising a substitution or deletion as compared to a     wild-type CD44 intracellular domain, or comprises or is a soluble     receptor comprising a CD44 extracellular domain that binds to     SIGLEC-15 of a certain cell. -   102. The modified cell of embodiment 101, wherein an inhibitory     effect of SIGLEC-15 on cytokine production of the human T cells of     the population is less than an inhibitory effect of SIGLEC-15 on     cytokine production of human T cells that do not comprise at least a     part of the nucleic acid sequence that encodes the modified CD44. -   103. The modified cell of any of preceding embodiments, wherein the     modified cell is engineered to express and secrete a therapeutic     agent such as a cytokine. -   104. The modified cell of embodiment 103, wherein the therapeutic     agent is or comprises IFN-γ. -   105. The modified cell of embodiment 103, wherein the therapeutic     agent is or comprises at least one of IL-6 or IFN-γ, IL-17, and     CCL19. -   106. The modified cell of embodiment 103, wherein the therapeutic     agent is or comprises IL-15 or IL-12, or a combination thereof. -   107. The modified cell of embodiment 103, wherein the therapeutic     agent is or comprises a recombinant or native cytokine. -   108. The modified cell of embodiment 103, wherein the therapeutic     agent comprises an FC fusion protein associated with a small     protein. -   109. The modified cell of embodiment 108, wherein the small protein     is or comprises IL-12, IL-15, IL-6 or IFN-γ. -   110. The modified cell of embodiment 103, wherein the therapeutic     agent is regulated by Hif1a, NFAT, FOXP3, and/or NFkB. -   111. The modified cell of embodiment 108, wherein the small protein     or the therapeutic agent is or comprises two or more recombinant or     native cytokines are collected via 2A or /IRES component. -   112. The nucleic acid, modified T cell or the method of any suitable     preceding embodiments, wherein the modified T cell comprises a first     targeting vector and a second targeting vector, the first targeting     vector comprising a nucleic acid sequence encoding a CAR binding a     blood antigen and the therapeutic agent, and the second targeting     vector comprises a nucleic acid sequence encoding a CAR biding solid     tumor antigen and a dominant negative form of the immune checkpoint     molecule. -   113. The nucleic acid, modified T cell or the method of any suitable     preceding embodiments, wherein the modified T cell comprises a first     targeting vector and a second targeting vector, the first targeting     vector comprising a nucleic acid sequence encoding a CAR binding     CD19 and the therapeutic agent, and the second targeting vector     comprises a nucleic acid sequence encoding a CAR biding UPK2, ACPP,     SIGLEC-15 or KISS1R and a dominant negative form of CD44. -   114. The nucleic acid, modified T cell or the method of any suitable     preceding embodiments, wherein the modified T cell comprises a first     targeting vector and a second targeting vector, the first targeting     vector comprising a nucleic acid sequence encoding a CAR binding a     blood antigen, and the second targeting vector comprises a nucleic     acid sequence encoding a CAR biding solid tumor antigen. -   115. The nucleic acid, modified T cell or the method of any suitable     preceding embodiments, wherein the modified T cell comprises a first     targeting vector and a second targeting vector, the first targeting     vector comprising a nucleic acid sequence encoding a CAR binding a B     cell antigen, and the second targeting vector comprises a nucleic     acid sequence encoding a CAR biding solid tumor antigen. -   116. The nucleic acid, modified T cell or the method of any suitable     preceding embodiments, wherein the solid tumor antigen is at least     one of antigens listed in Table 1, and/or the B cell antigen is     CD19, CD20, CD22, or BCMA. -   117. The nucleic acid, modified T cell or the method of any suitable     preceding embodiments, wherein the solid tumor antigen comprises at     least one of antigens listed in Table 1. -   118 A method of enhancing T cell expansion in a subject in need     thereof, the method comprising administering an effective amount of     the composition of T cells of any suitable preceding embodiments to     the subject, the subject having a higher level of T cell expansion     as compared with a subject that is administered an effective amount     of the CAR T cells that do not have the CAR binding the B cell     antigen. -   119. The nucleic acid, modified T cell or the method any suitable     preceding embodiments, wherein the modified T cell comprises a     nucleic acid sequence encoding hTERT, SV40LT, or a combination     thereof. -   120. The nucleic acid, modified T cell or the method of any suitable     preceding embodiments, wherein the modified T cell is more     proliferable than T cells without nucleic acid sequence. -   121. The isolated nucleic acid sequence, modified T cell or the     method of any suitable preceding embodiments, wherein the     proliferable cell remains functions of normal T cells/CAR T cells     such as cell therapy functions. -   122. The nucleic acid, modified T cell or the method any suitable     preceding embodiments, wherein the T cell comprises a CAR and is     cultured in the presence of an agent that is recognized by the     extracellular domain of the CAR, thereby producing a modified CAR     cell. -   123. The nucleic acid, modified T cell or the method of any suitable     preceding embodiments, wherein the integration of the nucleic acid     sequence encoding hTERT, the nucleic acid encoding SV40LT, or a     combination thereof includes genomic integration of the nucleic acid     sequence encoding hTERT, a nucleic acid encoding SV40LT, or a     combination thereof and constitutive expression of hTERT, SV40LT, or     a combination thereof. -   124. The nucleic acid sequence, modified T cell or the method of any     suitable preceding embodiments, wherein expression of hTERT, SV40LT,     or a combination thereof, is regulated by an inducible expression     system such as a rtTA-TRE system. -   125. The nucleic acid, modified T cell or the method of any suitable     preceding embodiments, wherein modified T cell comprises a nucleic     acid sequence encoding a suicide gene such as a an HSV-TK system. -   126. The nucleic acid, modified T cell or the method of any suitable     preceding embodiments, wherein the cell has a reduced     graft-versus-host disease (GVHD) response in a bioincompatible human     recipient as compared to the GVHD response of the primary human T     cell. -   127. The nucleic acid, modified T cell or the method of any suitable     preceding embodiments, wherein the cell has reduced expression of     endogenous TRAC gene. -   128. A fusion protein comprising an extracellular domain, a     transmembrane domain, and an intracellular domain, the extracellular     domain derived from a first molecule, the intracellular domain     derived from a second molecule, the first molecule different from     the second molecule. -   129. A fusion protein comprising an extracellular domain, a     transmembrane domain, and an intracellular domain, the extracellular     domain derived from a first molecule, the intracellular domain     derived from a second molecule, the first molecule different from     the second molecule, wherein the first molecule comprises a     co-stimulatory domain, and the second molecule comprises a cytokine     receptor. -   130. An isolated nucleic acid encoding the fusion protein of     embodiments 128 or 129. -   131. A modified cell comprising the isolated nucleic acid of     embodiment 130 or the fusion protein of embodiments 128 or 129. -   132. A pharmaceutical composition comprising a population of the     modified cells of embodiment 131. -   133. A method of causing or promoting a T cell response in a subject     in need thereof and/or treating a tumor of the subject, the method     comprising administering an effective amount of the composition of     embodiment 132 to the subject. -   134. The method of embodiment 133, further comprising: administering     an effective amount of an agonist antibody binding the extracellular     domain. -   135. The method of embodiment 134, wherein the binding of the     extracellular domain with the agnostic antibody causes recruitment     of multiple first molecules into a receptor oligomer in which     signaling activation of the second molecule is triggered or     enhanced. -   136. The fusion protein, isolated nucleic acid, modified cell,     pharmaceutical composition, or method of one of embodiments 128-135,     wherein the extracellular domain binds to an agonist antibody, and     wherein the agonist antibody is CDX 1140, SEA CD40, RO7009789, JNJ     64457107 (ADC1013), APX 005M, Chi Lob 7/4, TRX 518, MK 4166, MK     1248, GWN 323, INCAGN01876, BMS 986156, AMG 228, Tavolimab     (MED10562), PF 04518600, BMS 986178, MOXR 0916, GSK 3174998,     INCAGN01949, Utomilumab (PF 05082566), Urelumab (BMS 663513), GSK     3359609, JTX 2011, Theralizumab (TAB 08). -   137. The fusion protein, isolated nucleic acid, modified cell,     pharmaceutical composition, or method of one of embodiments 128-135,     wherein the extracellular domain is an extracellular domain of a     co-stimulatory molecule. -   138. The fusion protein, isolated nucleic acid, modified cell,     pharmaceutical composition, or method of one of embodiments 128-135,     wherein the extracellular domain is an extracellular domain of OX40,     CD40, 4-1BB, GITR, ICOS, CD28, CD27, HER2, or EGFR. -   139. The fusion protein, isolated nucleic acid, modified cell,     pharmaceutical composition, or method of one of embodiments 128-135,     wherein: the extracellular domain is an extracellular domain of     CD40, GITR, OX40, 41BB, ICOS, or CD28; -   the co-stimulatory molecule is costimulatory molecule selected from     the group consisting of CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1,     ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7,     LIGHT, NKG2C, B7-H3, and any combination thereof; and/or the     intracellular is an intracellular domain of a cytokine receptor. -   140. The fusion protein, isolated nucleic acid, modified cell,     pharmaceutical composition, or method of one of embodiments 128-135,     wherein the second molecule is OX40, CD40, 4-1BB, GITR, ICOS, CD28,     CD27, HER2, EGFR, IL10R, IL12R, IL18R1, IL23R, GP130, or IL15Ra. -   141. The fusion protein, isolated nucleic acid, modified cell,     pharmaceutical composition, or method of one of embodiments 128-135,     wherein the intracellular domain is an intracellular domain of     IL12R, IL18R1, IL23R, GP130, or IL15Ra. -   142. The fusion protein, isolated nucleic acid, modified cell,     pharmaceutical composition, or method of one of embodiments 128-141,     wherein the transmembrane domain is an intracellular domain of OX40,     CD40, 4-1BB, GITR, ICOS, CD28, CD27, HER2, EGFR, IL12R, IL18R1,     IL23R, GP130, or IL15Ra. -   143. The modified cell, pharmaceutical composition, or method of one     of embodiments 131-135, wherein the modified cell is lymphocyte,     leukocyte, or PBMC; or cells, NK cells, T cells, or dendritic cells. -   144. The modified cell, pharmaceutical composition, or method of one     of embodiments 131-143, wherein the modified cell further comprises     a Chimeric antigen receptor (CAR) or a modified TCR. -   145. The modified cell, pharmaceutical composition, or method of     embodiment 144, wherein the TCR is a modified TCR. -   146. The modified cell, pharmaceutical composition, or method of     embodiment 144, wherein the TCR is derived from spontaneously     occurring tumor-specific T cells in patients. -   147. The modified cell, pharmaceutical composition, or method of     embodiment 144, wherein the TCR binds a tumor antigen. -   148. The modified cell, pharmaceutical composition, or method of     embodiment 147, wherein the tumor antigen comprises CEA, gp100,     MART-1, p53, MAGE-A3, or NY-ESO-1, or the TCR comprises TCRγ and     TCRδ Chains or TCRα and TCRβ chains, or a combination thereof. -   149. The modified cell, pharmaceutical composition, or method of     embodiment 144, wherein the CAR comprises an extracellular domain, a     transmembrane domain, and an intracellular domain, and wherein the     extracellular domain binds an antigen. -   150. The modified cell, pharmaceutical composition, or method of     embodiment 149, wherein the intracellular domain comprises a     costimulatory signaling region that comprises an intracellular     domain of a costimulatory molecule selected from the group     consisting of CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS,     lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT,     NKG2C, B7-H3, and any combination thereof. -   151. The modified cell, pharmaceutical composition, or method of     embodiment 150, wherein the antigen is Epidermal growth factor     receptor (EGFR), Variant III of the epidermal growth factor receptor     (EGFRvIII), Human epidermal growth factor receptor 2 (HER2),     Mesothelin (MSLN), Prostate-specific membrane antigen (PSMA),     Carcinoembryonic antigen (CEA), Disialoganglioside 2 (GD2),     Interleukin-13Ra2 (IL13Rα2), Glypican-3 (GPC3), Carbonic anhydrase     IX (CAIX), L1 cell adhesion molecule (L1-CAM), Cancer antigen 125     (CA125), Cluster of differentiation 133 (CD133), Fibroblast     activation protein (FAP), Cancer/testis antigen 1B (CTAG1B), Mucin 1     (MUC1), Folate receptor-α (FR-α), CD19, FZD10, TSHR, PRLR, Muc 17,     GUCY2C, CD207, CD3, CD5, B-Cell Maturation Antigen (BCMA), or CD4. -   152. The modified cell, pharmaceutical composition, or method of one     of embodiments 131-151, wherein the modified cell or the T cells     comprise an additional CAR binding a solid tumor antigen, and the     CAR binds an antigen of a white blood cell. -   153. The modified cell, pharmaceutical composition, or method of one     of embodiments 131-151, wherein the modified cell or the T cells     comprise a dominant negative PD-1. -   154. The modified cell, pharmaceutical composition, or method of one     of embodiments 131-151, wherein the modified cell or the T cells     comprise a modified PD-1 lacking a functional PD-1 intracellular     domain. -   155. The modified cell, pharmaceutical composition or method of one     of embodiments 131-154, wherein the modified cell further comprises     a nucleic acid encoding therapeutic agent. -   156. The modified cell, pharmaceutical composition, or method of     embodiment 155, wherein the isolated nucleic acid comprises a     promoter comprising a binding site for a transcription modulator     that modulates the expression and/or secretion of the therapeutic     agent in the cell. -   157. The modified cell, pharmaceutical composition, or method of     embodiment 156, wherein the transcription modulator is or includes     Hif1a, NFAT, FOXP3, and/or NFkB. -   158. The modified cell, pharmaceutical composition, or method of     embodiment 157, wherein the promoter is responsive to the     transcription modulator. -   159. The modified cell, pharmaceutical composition, or method of     embodiment 157, wherein the promoter is operably linked to the     nucleic acid encoding the therapeutic agent such that the promoter     drives expression and/or secretion of the therapeutic agent in the     cell. -   160. The modified cell, pharmaceutical composition, or method of     embodiment 157, wherein expression of the therapeutic agent is     regulated by an inducible gene expression system. -   161. The modified cell, pharmaceutical composition, or method of     embodiment 160, wherein the inducible gene expression system     comprises or is a lac system, a tetracycline system, or a galactose     system. -   162. The modified cell, pharmaceutical composition, or method of     embodiment 160, wherein the inducible gene expression system     comprises or is a tetracycline system. -   163. The modified cell, pharmaceutical composition, or method of     embodiment 162, wherein the inducible gene expression system     comprises or is a tetracycline on system, and an inducer is     tetracycline, doxycycline, or an analog thereof. -   164. The modified cell, pharmaceutical composition, or method of one     of embodiments 131-163, wherein the modified cell is a T cell     derived from a primary human T cell isolated from a human donor. -   165. The modified cell, pharmaceutical composition, or method of     embodiment 164, wherein the cell has a reduced expression of     endogenous TRAC gene. -   166. The modified cell, pharmaceutical composition, or method of one     of embodiments 131-163, wherein the modified cell is a T cell     derived from a primary human T cell isolated from a subject having     cancer.

The related sequences are provided in Table 2 as well as PCT Patent Applications Nos: PCT/CN2016/075061, PCT/CN2018/08891, and PCT/US19/13068, which are incorporated herein by reference in their entirety.

EXAMPLES

Lentiviral vectors that encode individual CAR molecules were generated and transfected into T cells, which are elaborated below. Techniques related to cell cultures, construction of cytotoxic T lymphocyte assay can be found in “Control of large, established tumor xenografts with genetically retargeted human T cells containing CD28 and CD137 domains,” PNAS, Mar. 3, 2009, vol. 106, no. 9, 3360-3365 and “Chimeric Receptors Containing CD137 Signal Transduction Domains Mediate Enhanced Survival of T Cells and Increased Antileukemic Efficacy In Vivo,” Molecular Therapy, August 2009, vol. 17, no. 8, 1453-1464, which are incorporated herein by reference in their entirety. FIG. 6 shows flow cytometry results of T cells comprising a polynucleotide encoding CD19CAR (scFv binding CD19: SEQ ID NO: 5) and CD19CAR-2a-DNM1L (SEQ ID NO: 118) that have been cultured to Day 5. Sequence information corresponding to various vectors in the Examples are provided in Table 2.

FIGS. 7-10 show CAR expression and functions of T cells comprising a CAR and T cells comprising a CAR and secretable SIGLEC-15 scFv. T cells from various donors were obtained and transfected with vectors comprising CD19 CAR (SEQ ID NO: 5) or CD19 CAR-anti-SIGLEC-15 scFvs (SEQ ID NO: 115 or 116). As shown in FIG. 7, transduction of polynucleotides encoding anti-SIGLEC-15 scFvs did not affect the expression of CAR, which indicates that CAR expression is normal. Further, as shown in FIG. 8, T cells comprising polynucleotides encoding anti-SIGLEC-15 SCFV-1 and SCFV-2 have significant mRNA expression compared to control.

FIG. 9 shows flow cytometry results of the gene expression of tumor cells. The tumor cells comprising SIGLEC-15 and CD19+ does not express SIGLEC-15, while the tumor cells comprising CD19+ and SIGLEC-15+ express SIGLEC-15. FIG. 10 shows the killing function assay of T cells comprising CD19CAR and T cells comprising CD19CAR and secretable SIGLEC-15 scFv. Under different E:T ratios, various CART cells were co-cultured with SIGLEC-15-CD19+ and SIGLEC-15+-CD19+ tumor cells, and the function was determined by flow cytometry. As shown in FIG. 10, T cells comprising CD19 CAR and secretable SIGLEC-15 scFv showed enhanced killing of CD19+ and SIGLEC15+ tumor cells than T cells comprising CD19 CAR alone. These results demonstrate that secretable anti-SIGLEC-15 scFv blocked the SIGLEC-15-CD44 pathway to enhance the T cells' function.

All publications, patents and patent applications cited in this specification are incorporated herein by reference in their entireties as if each individual publication, patent or patent application were specifically and individually indicated to be incorporated by reference. While the foregoing has been described in terms of various embodiments, the skilled artisan will appreciate that various modifications, substitutions, omissions, and changes may be made without departing from the spirit thereof. 

The invention claimed is:
 1. A modified cell comprising a polynucleotide encoding an antibody binding SIGLEC-15, wherein the antibody is secretable or attached to the membrane of the modified cell, wherein the polynucleotide comprises a polynucleotide encoding at least one of SEQ ID NOS: 115 and
 116. 2. The modified cell of claim 1, wherein the modified cell further comprises a dominant negative form of an immune checkpoint inhibitor selected from the group consisting of programmed death 1 (PD-1), cytotoxic T lymphocyte antigen-4 (CTLA-4), B- and T-lymphocyte attenuator (BTLA), T cell immunoglobulin mucin-3 (TIM-3), lymphocyte-activation protein 3 (LAG-3), T cell immunoreceptor with Ig and ITIM domains (TIGIT), leukocyte-associated immunoglobulin-like receptor 1 (LAIRD, CD44, natural killer cell receptor 2B4 (2B4), and CD
 160. 3. The modified cell of claim 1, wherein the modified T cell is engineered to express and secrete a therapeutic agent.
 4. The modified cell of claim 3, wherein the therapeutic agent comprises at least one of IL-33, IL-1β, TNFα, MALP-2, IL1, IL17, G-CSF or GM-CSF, IL-6, IL-12, and IFN-γ.
 5. The modified cell of claim 3, wherein the therapeutic agent is regulated by Hif1a, NFAT, FOXP3, and/or NFkB.
 6. The modified cell of claim 1, wherein the modified cell comprises an antigen-binding molecule binding a tumor antigen, and the modified cell comprises a T cell derived from a healthy donor or a subject having cancer.
 7. The modified cell of claim 6, wherein the antigen-binding molecule is a chimeric antigen receptor (CAR), which comprises an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain.
 8. The modified cell of claim 7, wherein the antigen-binding domain binds a tumor antigen selected from a group consisting of: TSHR, CD19, CD123, CD22, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, Mesothelin, IL-11Ra, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-beta, SSEA-4, CD20, Folate receptor alpha, ERBB2 (Her2/neu), MUC1, EGFR, NCAM, Prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gp100, bcr-abl, tyrosinase, EphA2, Fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, Folate receptor beta, TEM1/CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, Polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a, MAGE-A1, legumain, HPV E6, E7, MAGE A1, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostein, surviving, telomerase, PCTA-1/Galectin 8, MelanA/MART1, Ras mutant, hTERT, sarcoma translocation breakpoints, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, Androgen receptor, Cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxyl esterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, and IGLL1.
 9. The modified cell claim 8, wherein the intracellular signaling domain comprises a co-stimulatory signaling domain, or a primary signaling domain and a co-stimulatory signaling domain, wherein the co-stimulatory signaling domain comprises a functional signaling domain of a protein selected from the group consisting of CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, CD5, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, NKp44, NKp30, NKp46, and NKG2D.
 10. The modified cell of claim 6, wherein the antigen-binding molecule is a modified T cell receptor (TCR).
 11. The modified cell of claim 10, wherein the TCR is derived from spontaneously occurring tumor-specific T cells in patients.
 12. The modified cell of claim 11, wherein the TCR binds to a tumor antigen.
 13. The modified cell of claim 12, wherein the tumor antigen comprises CEA, gp100, MART-1, p53, MAGE-A3, or NY-ESO-1.
 14. The modified cell of claim 12, wherein the TCR comprises TCRγ and TCRδ chains, TCRα and TCRβ chains, or a combination thereof.
 15. The modified cell of claim 1, wherein the cell is an immune cell.
 16. The modified cell of claim 15, wherein the immune cell is a T cell or an NK cell.
 17. The modified cell of claim 16, wherein the immune cell is a CD4+ T cell, a CD8+ T cell, or a combination thereof.
 18. The modified cell of claim 3, wherein the therapeutic agent is a cytokine.
 19. The modified cell of claim 1, wherein the polynucleotide further comprises at least one of SEQ ID NO: 117, 118, and
 119. 20. A modified cell comprising a polynucleotide encoding at least one of SEQ ID NOS: 117, 118, and
 119. 21. The modified cell of claim 20, wherein the modified cell comprises an antigen-binding molecule binding a tumor antigen, and the modified cell comprises a T cell derived from a healthy donor or a subject having cancer.
 22. The modified cell of claim 21, wherein the antigen-binding molecule is a CAR or a modified TCR. 